WO1997037676A1 - Nouvelles proteines de salive d'ectoparasite et appareil pour les recueillir - Google Patents

Nouvelles proteines de salive d'ectoparasite et appareil pour les recueillir Download PDF

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Publication number
WO1997037676A1
WO1997037676A1 PCT/US1997/005959 US9705959W WO9737676A1 WO 1997037676 A1 WO1997037676 A1 WO 1997037676A1 US 9705959 W US9705959 W US 9705959W WO 9737676 A1 WO9737676 A1 WO 9737676A1
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Prior art keywords
seq
lys
glu
nucleic acid
leu
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PCT/US1997/005959
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English (en)
Inventor
Shirley Wu Hunter
Gek-Kee Sim
Eric R. Weber
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Heska Corporation
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Application filed by Heska Corporation filed Critical Heska Corporation
Priority to US09/171,156 priority Critical patent/US6368846B1/en
Priority to JP53649997A priority patent/JP4694657B2/ja
Priority to CA2250835A priority patent/CA2250835C/fr
Priority to AU24531/97A priority patent/AU719742B2/en
Priority to EP97920304A priority patent/EP0939642A4/fr
Priority to AU49849/97A priority patent/AU4984997A/en
Priority to US08/981,799 priority patent/US6576238B1/en
Priority to PCT/US1997/018669 priority patent/WO1998045408A2/fr
Publication of WO1997037676A1 publication Critical patent/WO1997037676A1/fr
Priority to US09/004,730 priority patent/US6485968B1/en
Priority to US11/694,771 priority patent/US20080045693A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/22—Devices for withdrawing samples in the gaseous state
    • G01N1/24—Suction devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/4359—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from fleas
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to a noveJ product and method for isolating ectoparasite saliva proteins, and a novel product and method for detecting and/or treating allergic dermatitis in an animal.
  • hypersensitive responses to fleabites is manifested m a disease called flea allergy dermatitis (FAD) .
  • FAD flea allergy dermatitis
  • Hypersensitivity refers to a state of altered reactivity in which an animal, having been previously exposed to a compound, exhibits an allergic response to the compound upon subsequent exposures.
  • Hypersensitive responses include immediate and delayed-type hypersensitivity, and in particular Type I, Type II, Type III and Type IV hypersensitivities (described in detail in Janeway et al., Ixn unobiology, Garland Publishing, New York, 1994, which is incorporated in its entirety by this reference) .
  • allergens Foreign compounds that induce symptoms of immediate and/or delayed hypersensitivity are herein referred to as allergens.
  • allergen primarily refers to foreign compounds capable of causing an allergic response.
  • the term can be used interchangeably with the term "antigen,” especially with respect to a foreign compound capable of inducing symptoms of immediate and/or delayed hypersensitivity.
  • Factors that influence an animal's susceptibility to an allergen can include a genetic component and/or environmental exposure to an allergen. Animals can be de-sensitized to an allergen by repeated injections of the allergen to which an animal is hypersensitive .
  • FAD can have manifestations of both immediate and delayed-type hypersensitivity (described m detail in Janeway et al . , ibid. ) .
  • Effective treatment of FAD has been difficult if not impossible to achieve.
  • FAD afflicts about 15% of cats and dogs in flea endemic areas and the frequency is increasing each year. In a geographical area, effective flea control requires treatment of all animals.
  • One treatment investigators have proposed includes desensitization of animals using flea allergens. However, reliable, defined preparations of flea allergens are needed for such treatments.
  • flea allergens responsible for FAD had not been clearly defined. Whole flea antigen preparations have been used to diagnose and desensitize animals with FAD
  • the allergenic factors of flea saliva have characterized the allergenic factors of flea saliva as being haptens having molecular weights of less than 6 kilodaltons (kD) . That they are not proteins is also supported by the finding that they are not susceptible to degradation when exposed to strong acids (e.g., 6 N hydrochloric acid) or heat. Some of the particular low molecular weight allergenic factors have also been characterized as being a highly fluorescent aromatic fraction (Young et al., ibi d. ) . In addition, studies by such investigators have indicated that m order to be allergenic, such factors need to be associated with adjuvants and/or carriers, such as collagen or portions of the membrane used to collect the oral secretions. Moreover, the methods described to collect flea saliva factors were difficult and unpredictable. Furthermore the factors isolated by these methods were typically contaminated with material from the fleas, their culture medium or the skm- based membranes used to allow the fleas to feed.
  • One embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent conditions with a gene including a flea saliva gene comprising a nucleic acid sequence including SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID N0*.71, SEQ ID N0:73, SEQ ID NO:74, SEQ ID N0:76 and a nucleic acid sequence encoding an ammo acid sequence selected from the group consisting of SEQ ID NO: 78 and SEQ ID NO: 87.
  • the present invention also includes a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 87.
  • Another embodiment of the present invention includes an isolated protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an ammo acid sequence including SEQ ID NO:53, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO: 78 and SEQ ID NO: 87.
  • Also included in the present invention are recombinant molecules and cells having a nucleic acid molecule of the present invention.
  • Another aspect of the present invention includes an antibody capable of selectively binding to an ectoparasite protein, or mimetope.
  • a therapeutic composition for treating allergic dermatitis comprising a formulation comprising at least one isolated ectoparasite saliva protein, wherein said ectoparasite saliva protein comprises at least a portion of an ammo acid sequence, where said portion is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence including SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:76 and
  • a preferred therapeutic composition of the present invention also includes an excipient, an adjuvant and/or a carrier. Also included in the present invention is a method to desensitize a host animal to allergic dermatitis. The method includes the step of administering to the animal a therapeutic composition of the present invention. Other embodiments of the present invention include methods to identify an animal susceptible to or having allergic dermatitis, using m vi vo or m vi tro methods.
  • an animal susceptible to or havmg allergic dermatitis is identified m vi vo by the method comprising: (a) administering to a site on the animal a formulation comprising at least one isolated ectoparasite saliva protein, m which the ectoparasite saliva protein comprises an ammo acid sequence including SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 87; and (b) comparing a reaction resulting from administration of the formulation with a reaction resulting from administration of a control solution, in which the animal is determined to be susceptible to or to have allergic dermatitis if the reaction to the formulation is at least as large as said reaction to the positive control solution, and in which the animal is determined not to be susceptible to or not to have allergic dermatitis if the reaction to the formulation is about the same size as said reaction to the
  • an animal susceptible to or having allergic dermatitis is identified m vi tro by measuring the presence of antibodies indicative of allergic dermatitis m the animal using the method comprising: (a) contacting a formulation with a body fluid from an animal under conditions sufficient for formation of an immunocomplex between the formulation and the antibodies, if present, in the body fluid, the formulation comprising at least one isolated ectoparasite saliva protein, in which the ectoparasite saliva protein comprises an ammo acid sequence including SEQ ID NO:53, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 87; and (b) determining the amount of lmmunocomplex formed, which formation of the immunocomplex indicates that the animal is susceptible to or has allergic dermatitis.
  • the present invention further relates to an assay kit for testing if an animal is susceptible to or has allelic dermatitis, the kit comprising: (a) a formulation comprising at least one isolated ectoparasite saliva protein, in which the ectoparasite saliva protein comprises an amino acid sequence including SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO: 87; and (b) a means for determining if the animal is susceptible to or has allergic dermatitis, in which the means comprises use of the formulation to identify animals susceptible to or having allergic dermatitis.
  • the present invention includes a novel product and method for diagnosing and treating allergic dermatitis of animals to ectoparasites.
  • ectoparasites are external living parasites that attach and feed through the sk n of a host animal. Ectoparasites include parasites that live on a host animal and parasites that attach temporarily to an animal m order to feed. Also, according to the present invention, ectoparasite saliva refers to the material released from the mouth of an ectoparasite when the ectoparasite attempts to feed in response to a temperature differential. Ectoparasite saliva includes ectoparasite saliva products.
  • One embodiment of the present invention is a formulation that contains ectoparasite saliva products that can be used to diagnose and/or treat animals susceptible to or having (i.e., suffering from) allergic dermatitis.
  • Preferred types of allergic dermatitis to diagnose and/or treat using ectoparasite saliva products of the present invention include flea allergy dermatitis, Culi coides allergy dermatitis and mosquito allergy dermatitis.
  • a preferred type of allergic dermatitis to diagnose and/or treat using ectoparasite saliva products of the present invention is flea allergy dermatitis.
  • an animal that is susceptible to allergic dermatitis refers to an animal that is genetically pre-disposed to developing allergic dermatitis and/or to an animal that has been primed with an antigen such a manner that re-exposure to the antigen results in symptoms of allergy that can be perceived by, for example, observing the animal or measuring antibody production by the animal to the antigen.
  • animals susceptible to allergic dermatitis can include animals having sub-clinical allergic dermatitis.
  • SuD-climcal allergic dermatitis refers to a condition m which allergy symptoms cannot be detected by simply observing an animal (i.e., manifestation of the disease can include the presence of anti-ectoparasite saliva protein antibodies withm an affected animal but no dermatitis) .
  • sub-clinical allergic dermatitis can be detected using in vi vo or m vi tro assays of the present invention, as described in detail below.
  • Reference to animals having allergic dermatitis includes animals that do display allergy symptoms that can be detected by simply observing an animal and/or by using m vivo or m vi tro assays of the present invention, as described in detail below.
  • One embodiment of the present invention is a formulation that includes one or more isolated ectoparasite saliva proteins.
  • an isolated protein is a protein that has been removed from its natural milieu.
  • An isolated ectoparasite saliva protein can, for example, be obtained from its natural source, be produced using recombinant DNA technology, or be synthesized chemically.
  • an isolated ectoparasite saliva protein can be a full-length ectoparasite saliva protein or any homologue of such a protein, such as an ectoparasite saliva protein in which ammo acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivat zed (e.g., by glycosylation, phosphorylation, acetylation, myristylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl mositol) .
  • ammo acids e.g., a truncated version of the protein, such as a peptide
  • derivat zed e.g., by glycosylation, phosphorylation, acetylation, myristylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl
  • a homologue of an ectoparasite saliva protein is a protein having an amino acid sequence that is sufficiently similar to a natural ectoparasite saliva protein ammo acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing under stringent conditions to (i.e., with) a nucleic acid molecule encoding the natural ectoparasite saliva protein (i.e., the complement of a nucleic acid sequence encoding the natural ectoparasite saliva protein am o acid sequence) .
  • a nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i.e., can form a complete double helix with) the strand for which the sequence is cited. It is to be noted that a double-stranded nucleic acid molecule of the present invention for which a nucleic acid sequence has been determined for one strand that represented by a SEQ ID NO also comprises a complementary strand havmg a sequence that is a complement of that SEQ ID NO.
  • nucleic acid molecules of the present invention which can be either double-stranded or smgle- stranded, include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with either a given SEQ ID NO denoted herem and/or with the complement of that SEQ ID NO, which may or may not be denoted herein. Methods to deduce a complementary sequence are known to those skilled in the art.
  • stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules, including oligonucleotides, are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Labs Press, 1989; Sambrook et al., ibi d. , is incorporated by reference herem in its entirety. Stringent hybridization conditions typically permit isolation of nucleic acid molecules havmg at least about 70% nucleic acid sequence identity with the nucleic ac d molecule being used to probe in the hybridization reaction.
  • the minimal size of a protein homologue of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein.
  • the size of the nucleic acid molecule encoding such a protein homologue is dependent on nucleic acid composition and percent homology between the nucleic acid molecule and complementary sequence as well as upon hybridization conditions per se (e.g., temperature, salt concentration, and formamide concentration) .
  • the minimal size of such nucleic acid molecules is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 17 bases m length if they are AT- ⁇ ch.
  • the minimal size of a nucleic acid molecule used to encode an ectoparasite saliva protein homologue of the present invention is from about 12 to about 18 nucleotides length.
  • the maximal size of such a nucleic acid molecule that the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes, or portions thereof.
  • an ectoparasite saliva protein homologue of the present invention is from about 4 to about 6 ammo acids in length, with preferred sizes depending on whether a full-length, multivalent (i.e., fusion protein having more than one domain each of which has a function) , or functional portions of such proteins are desired.
  • Ectoparasite saliva protein homologues can be the result of allelic variation of a natural gene encoding an ectoparasite saliva protein.
  • a natural gene refers to the form of the gene found most often m nature.
  • Ectoparasite saliva protein homologues can be produced using techniques known in the art including, but not limited to, direct modifications to a gene encoding a protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
  • Preferred ectoparasite saliva proteins of the present invention are capable of detecting and/or treating allergic dermatitis resulting from the bites of ectoparasites.
  • a preferred ectoparasite saliva protein homologue includes at least one epitope capable of eliciting a hypersensitive response to the natural ectoparasite saliva protein counterpart.
  • An ectoparasite saliva protein homologue can also include an epitope capable of hyposensitiz g an animal to the natural form of the protein.
  • an ectoparasite saliva protein homologue to detect and/or treat (i.e., immunomodulate or regulate by, for example, desensitizing) the hypersensitivity of an animal susceptible to or having allergic dermatitis, can be tested using techniques known to those skilled m the art. Such techniques include skm tests and immunoabsorbent assays as described m detail below. Additional preferred ectoparasite saliva proteins of the present invention have other activities that include activities important for feeding and survival of the ectoparasite.
  • a formulation of the present invention can comprise a protein having at least a portion of an isolated ectoparasite saliva protein.
  • "at least a portion of an ectoparasite saliva protein” refers to a portion of an ectoparasite saliva protein encoded by a nucleic acid molecule that is capable of hybridizing, under stringent conditions, with a nucleic acid encoding a full-length ectoparasite saliva protein of the present invention.
  • Preferred portions of ectoparasite saliva proteins are useful for detecting and/or treating allergic dermatitis resulting from the bites of ectoparasites. Additional preferred portions have activities important for flea feeding and survival. Suitable sizes for portions of an ectoparasite saliva protein of the present invention are as disclosed for saliva protein homologues of the present invention.
  • a formulation of the present invention can include saliva products from any ectoparasites.
  • More preferred ectoparasites from which to obtain saliva products include fleas; ticks, including both hard ticks of the family Ixodidae (e.g., Ixodes and AmJblyomma) and soft ticks of the family Argasidae (e.g., Orm thodoros, such as 0. parke ⁇ and 0.
  • flies such as midges (e.g., Culi coides) , mosquitos, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis-causmg flies and biting gnats; ants; spiders, lice; mites; and true bugs, such as bed bugs and kissing bugs, including those carrying Chagas disease.
  • midges e.g., Culi coides
  • ectoparasite saliva products include those from fleas, mosquitos, midqes, sandflies, blackflies, ticks and Rhodm us, with products from fleas, mosquitos and Culi coides being even more preferred.
  • a particularly preferred formulation of the present invention includes flea saliva proteins.
  • Preferred flea saliva products include those from Ctenocephali des, Xenopsylla, Pulex, Tunga, Nosopsyll us, Diamanus, Ctopsyll us and Echidnophaga fleas, with saliva products from Ctenocephalides cams and Ctenocephalides feli s fleas being even more preferred.
  • many of the following embodiments discuss flea saliva proteins. Such discussion of flea saliva proteins is not intended, in any way, to limit the scope of the present invention.
  • a formulation of the present invention includes at least a portion of an ectoparasite saliva protein homologue having at least a portion of one of the following ammo acid sequences: SEQ ID NO: 53, SEQ ID NO:62, SEQ ID NO: 65, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO: 78 and SEQ ID NO: 87 and/or other sequences disclosed herem.
  • a formulation of the present invention can include at least one isolated protein having (i.e., including) at least a portion of one of the ammo acid sequences identified m the Sequence ID Listing, and more specifically an ammo acid sequence selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 87.
  • ectoparasite saliva proteins of the present invention include, but are not limited to, full-length proteins, hybrid proteins, fusion proteins, multivalent proteins, and proteins that are truncated homologues of, or are proteolytic products of, at least a portion of a protein havmg at least a portion of one of the following ammo acid sequences: SEQ ID NO:53, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:87 and/or other sequences disclosed herein.
  • the term hybrid protein refers to a single protein produced from two different proteins.
  • a formulation of the present invention can include flea saliva proteins that have undergone post-translational modification. Such modification can include, for example, glycosylation.
  • Glycosylation can include addition of N-lmked and/or 0- linked oligosaccharides. It is to be appreciated that post-translational modification of a protein of the present invention can contribute to an epitope's ability to induce an allergic response against the protein m an immediate or delayed hypersensitivity response.
  • Another embodiment of the present invention is an isolated nucleic acid molecule capable of hybridizing, under stringent conditions, with an ectoparasite saliva protein gene encoding an ectoparasite saliva protein of the present invention.
  • an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) . As such, "isolated” does not reflect the extent to which the nucleic acid molecule has been purified.
  • An isolated nucleic acid molecule can include DNA, RNA, or derivatives of either DNA or RNA.
  • An isolated nucleic acid molecule of the present invention can be obtained from its natural source either as an entire (i.e., complete) gene or a portion thereof capable of forming a stable hybrid with that gene.
  • the phrase "at least a portion of" an entity refers to an amount of the entity that is at least sufficient to have the functional aspects of that entity.
  • at least a portion of a nucleic acid sequence is an amount of a nucleic acid sequence capable of forming a stable hybrid with the corresponding gene under stringent hybridization conditions.
  • isolated nucleic acid molecule of the present invention can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • Isolated ectoparasite saliva protein nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules m which nucleotides have been inserted, deleted, substituted, and/or inverted m such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode an ectoparasite saliva protein of the present invention or to form stable hybrids under stringent conditions with natural nucleic acid molecule isolates.
  • An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one ectoparasite saliva protein of the present invention, examples of such proteins being disclosed herein.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding an ectoparasite saliva protein.
  • ectoparasite saliva proteins of the present invention include, but are not limited to, proteins having full-length ectoparasite saliva protein coding regions, portions thereof, and other ectoparasite saliva protein homologues. It is to be appreciated that an ectoparasite saliva protein of the present invention can be encoded by a full- length nucleic acid sequence which encodes a polyprotem. The polyprotem can be post-translationally processed into multiple proteins which are found m saliva.
  • an ectoparasite saliva protein gene includes all nucleic acid sequences related to a natural ectoparasite saliva protein gene such as regulatory regions that control production of an ectoparasite saliva protein encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself.
  • a nucleic acid molecule of the present invention can be an isolated natural ectoparasite saliva protein nucleic acid molecule or a homologue thereof.
  • a nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof.
  • the minimal size of an ectoparasite saliva protein nucleic acid molecule of the present invention is the minimal size capable of forming a stable hybrid under stringent hybridization conditions with a corresponding natural gene.
  • nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
  • classic mutagenesis techniques and recombinant DNA techniques such as site-directed mutagenesis
  • chemical treatment of a nucleic acid molecule to induce mutations
  • restriction enzyme cleavage of a nucleic acid fragment ligation of nucleic acid fragments
  • PCR polymerase chain reaction
  • Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid (e.g., the ability of a homologue to elicit an allergic response in animals having allergic dermatitis or the ability of a homologue to act as an anti-coagulant) and/or by hybridization with isolated ectoparasite saliva protein nucleic acids under stringent conditions.
  • One embodiment of the present invention is an ectoparasite saliva protein nucleic acid molecule that encodes a protein havmg at least a portion of one or more of the following amino acid sequences: SEQ ID N0:1, as well as with the complements of any of these sequences or homologues thereof.
  • Such preferred nucleic acid molecules can hybridize to the coding and/or complementary strand.
  • a preferred nucleic acid molecule of the present invention is capable of hybridizing under stringent conditions to the coding strand and/or to the strand complementary to the coding strand of a nucleic acid molecule that encodes at least a portion of such a flea saliva protein or homologue thereof.
  • a particularly preferred nucleic acid sequence is a nucleic acid sequence having at least about 65 percent, preferably at least about 75 percent, more preferably at least about 85 percent, and even more preferably at least about 95 percent homology with a nucleic acid sequence encoding at least a portion of one or more of the following amino acid sequences .
  • nucleic acid molecules can be a full-length gene and/or a nucleic acid molecule encoding a full-length protein, a hybrid protein, a fusion protein, a multivalent protein or a truncation fragment. More preferred nucleic acid molecules of the present invention comprise isolated nucleic acid molecules having a nucleic acid sequence as represented by SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO: 76, a nucleic acid sequence encoding ammo acid sequence SEQ ID NO:78 or SEQ ID NO: 87, or other sequences disclosed herein.
  • SEQ ID NO: 52 a nucleic acid sequence that includes about 595 nucleotides of the apparent gene encoding flea saliva protein fspG5 (denoted nfspG5 ) , encodes a protein of about 90 amino acids (denoted as PfspG5 90 ) , represented by SEQ ID NO: 53.
  • the entire translation product of fspG5 is apparently about 71 amino acids and is denoted SEQ ID NO:56.
  • SEQ ID NO: 61 a nucleic acid sequence that includes about 1007 nucleotides of the apparent gene encoding flea saliva protein fspl (denoted nfspl 1007 ) r encodes a protein of about 155 amino acids (denoted Pfspl 155 ) , which is denoted SEQ ID NO: 62.
  • SEQ ID NO: 64 a nucleic acid sequence that includes about 1205 nucleotides of the apparent gene encoding flea saliva protein fspNS (denoted nfspN5 120b ) , encodes a protein of about 353 amino acids (denoted PfspN5 353 ) , which is denoted SEQ ID NO: 65.
  • SEQ ID NO: 71 a nucleic acid sequence that includes about 406 nucleotides of the apparent gene encoding a fspN6 flea saliva protein
  • SEQ ID NO: 72 a nucleic acid sequence that includes about 420 nucleotides of the apparent gene encoding a fspJ flea saliva protein, encodes a protein of about 72 amino acids, which is denoted SEQ ID NO:75.
  • nucleic acid molecule of an ectoparasite saliva protein of the present invention allows one skilled in the art to make copies of that nucleic acid molecule as well as to obtain a nucleic acid molecule including additional portions of ectoparasite saliva protein-encoding genes (e.g., nucleic acid molecules that include the translation start site and/or transcription and/or translation control regions), and/or ectoparasite saliva protein nucleic acid molecule homologues. Knowing a portion of an ammo acid sequence of an ectoparasite saliva protein of the present invention allows one skilled in the art to clone nucleic acid sequences encoding such an ectoparasite saliva protein.
  • a desired ectoparasite saliva protein nucleic acid molecule can be obtained m a variety of ways including screening appropriate expression libraries with antibodies which bind to ectoparasite saliva proteins of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries or DNA; and PCR amplification of appropriate libraries, or RNA or DNA using oligonucleotide primers of the present invention (genomic and/or cDNA libraries can be used) .
  • preferred cDNA libraries include cDNA libraries made from unfed whole flea, fed whole flea, fed flea idgut, unfed flea midgut, and flea salivary gland. Techniques to clone and amplify genes are disclosed, for example, m Sambrook et al., ibid. The Examples section includes examples of the isolation of cDNA sequences encoding flea saliva proteins of the present invention.
  • the present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention that encode at least a portion of one or more of the following am o acid sequences: SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO:70, SEQ ID NO: 72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:87, or homologues thereof, such oligonucleotides can hybridize to the coding or non-coding strand of a double-stranded nucleic acid molecule.
  • Certain preferred oligonucleotides are capable of hybridizing to nucleic acid molecules including nucleic acid sequences represented by SEQ ID NO: 52, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 71, SEQ ID NO:74, a nucleic acid sequence that encodes SEQ ID NO: 78 or SEQ ID NO: 87, or complements thereof.
  • Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either.
  • the minimal size of such oligonucleotides is the size required to form a stable hybrid between a given oligonucleotide and the complementary sequence on another nucleic acid molecule of the present invention. Minimal size characteristics are disclosed herein. The size of the oligonucleotide must also be sufficient for the use of the oligonucleotide m accordance with tne present invention.
  • Oligonucleotides of the present invention can be used m a variety of applications including, but not limited to, as probes to identify additional nucleic acid molecules, as primers to amplify or extend nucleic acid molecules or in therapeutic applications to inhibit, for example, expression of saliva proteins by ectoparasites.
  • Such therapeutic applications include the use of such oligonucleotides m, for example, antisense-, triplex formation-, ribozyme- and/or RNA drug- based technologies.
  • the present invention therefore, includes such oligonucleotides and methods to interfere with the production of ectoparasite saliva proteins by use of one or more of such technologies.
  • the present invention also includes a recombinant vector, which includes an ectoparasite saliva protein nucleic acid molecule of the present invention inserted into any vector capable of delivering the nucleic acid molecule into a host cell.
  • a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to ectoparasite saliva protein nucleic acid molecules of the present invention.
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
  • Recombinant vectors can be used the cloning, sequencing, and/or otherwise manipulating of ectoparasite saliva protein nucleic acid molecules of the present invention.
  • recombinant vector herein referred to as a recombinant molecule and described in more detail below, can be used in the expression of nucleic acid molecules of the present invention.
  • Preferred recombinant vectors are capable of replicating in the transformed cell.
  • a preferred nucleic acid molecule to include in a recombinant vector of the present invention is a nucleic acid molecule that encodes at least a portion of one or more of the following amino acid sequences: SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:87, or other sequences disclosed herein, or homologues thereof, and nucleic acid molecules including at least a portion of a nucleic acid sequence represented by SEQ ID NO: 52, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO:64, SEQ ID NO:71, SEQ ID NO: 74, a nucleic acid sequence that encodes SEQ ID NO: 78 or SEQ ID NO: 87, or other sequences disclosed herein, or complements thereof.
  • a more preferred sequences to include in a recombinant vector include nfspG5 595 , nfspG5 270 nfspG5, 13 , nfspl ]007 , nfspN5 1205 , nfspN5 10 ⁇ g nfspN6 406 and nfspJ 420 .
  • Preferred recombinant molecules of the present invention include pCro-nfspG5 3 and pCro-nfspl 4 ⁇ 4 , the production of which are described in detail in the Examples section.
  • an isolated ectoparasite saliva protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein.
  • a preferred cell to culture is a recombinant cell that is capable of expressing the ectoparasite saliva protein, the recombinant cell being produced by transforming a host cell with one or more nucleic acid molecules of the present invention.
  • Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microm ection, lipofection, adsorption, and protoplast fusion.
  • a recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.
  • Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites withm a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
  • Preferred nucleic acid molecules with which to transform a host cell include one or more nucleic acid molecules that are as disclosed herein for including m recombinant vectors of the present invention.
  • Suitable host cells to transform include any cell that can be transformed and that can express the introduced ectoparasite saliva protein. Such cells are, therefore, capable of producing ectoparasite saliva proteins of the present invention after being transformed with at least one nucleic acid molecule of the present invention.
  • Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule.
  • Suitable host cells of the present invention can include bacterial, fungal (including yeast), insect, animal and plant cells.
  • Preferred host cells include bacterial, yeast, insect and mammalian cells, with bacterial (e.g., E. coli ) and insect (e.g., Spodoptera ) cells being particularly preferred.
  • a recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences.
  • the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule.
  • the expression vector is also capable of replicating withm the host cell.
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including m bacterial, fungal, insect, animal, and/or plant cells.
  • nucleic acid molecules of the present invention can be operatively linked to expression vectors containing regulatory sequences such as promoters, operators, repressors, enhancers, termination sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention.
  • a transcription control sequence includes a sequence which is capable of controlling the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function m at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
  • Preferred transcription control sequences include those which function m bacterial, yeast, helminth, insect and mammalian cells, such as, but not limited to, tac, l ac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda ( ⁇ ) (such as ⁇ p L and ⁇ p k and fusions that include such promoters), bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionem, alpha mating factor, Pi chia alcohol oxidase, alphavirus subgenomic promoters (such as S dbis virus subgenomic promoters) , baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus 40, retrovirus actm, retroviral long terminal repeat, Rous
  • transcription control sequences include tissue-specific promoters and enhancers as well as lymphokme-mducible promoters (e.g., promoters inducible by interferons or mterleukms) .
  • Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with a DNA sequence encoding an ectoparasite saliva protein.
  • Expression vectors of the present invention may also contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed ectoparasite saliva protein to be secreted from the cell that produces the protein.
  • Suitable signal segments include an ectoparasite saliva protein signal segment or any heterologous signal segment capable of directing the secretion of an ectoparasite saliva protein, including fusion proteins, of the present invention.
  • Preferred signal segments include, but are not limited to, tissue plasmmogen activator (t- PA) , mterferon, mterleukm, growth hormone, histocompatibility and viral envelope glycoprotein signal segments .
  • Expression vectors of the present invention may also contain fusion sequences which lead to the expression of inserted nucleic acid molecules of the present invention as fusion proteins.
  • Inclusion of a fusion sequence as part of an ectoparasite nucleic acid molecule of the present invention can enhance the stability during production, storage and/or use of the protein encoded by the nucleic acid molecule.
  • a fusion segment can function as a tool to simplify purification of an ectoparasite saliva protein, such as to enable purification of the resultant fusion protein using affinity chromatography.
  • a suitable fusion segment can be a domain of any size that has the desired function (e.g., increased stability and/or purification tool) . It is withm the scope of the present invention to use one or more fusion segments.
  • Fusion segments can be joined to ammo and/or carboxyl termmi of an ectoparasite saliva protein.
  • Linkages between fusion segments and ectoparasite saliva proteins can be constructed to be susceptible to cleavage to enable straight-forward recovery of the ectoparasite saliva proteins.
  • Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid sequence that encodes a protein including the fusion segment attached to either the carboxyl and/or ammo terminal end of an ectoparasite saliva protein.
  • a recombinant molecule of the present invention is a molecule that can include at least one of any nucleic acid molecule heretofore described operatively linked to at least one of any transcription control sequence capable of effectalveoli regulating expression of the nucleic acid molecule (s) in the cell to be transformed.
  • a preferred recombinant molecule includes one or more nucleic acid molecules that are as disclosed herein for including in a recombinant vector of the present invention.
  • a recombinant cell of the present invention includes any cells transformed with at least one of any nucleic acid molecules of the present invention.
  • a preferred recombinant cell is a cell transformed with at least one nucleic acid molecule that encode a protein having at least a portion of one or more of the following ammo acid sequences: SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO: 78, SEQ ID NO: 87, or other sequences disclosed herem, or homologues thereof, and nucleic acid molecules including at least a portion of a nucleic acid sequence represented by SEQ ID NO:52, SEQ ID NO:58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 71, SEQ ID NO: 74, a nucleic acid sequence that encodes SEQ ID NO: 78 or SEQ ID NO: 87,
  • Particularly preferred recombinant cells include E. coli transformed with at least one of the aforementioned nucleic acid molecules.
  • Preferred recombinant cells of the present invention include E. coli :pCro-nfspG5 21 , and E. coli :pCro- nfspl 474 ,
  • recombinant DNA technologies can improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Sh e-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant protein production during fermentation.
  • the activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing the resultant protein.
  • recombinant cells can be used to produce an ectoparasite saliva protein of the present invention by culturing such cells under conditions effective to produce such a protein, and recovering the protein.
  • Effective conditions to produce a protein include, but are not limited to, appropriate media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • An appropriate, or effective, medium refers to any medium in which a cell of the present invention, when cultured, is capable of producing an ectoparasite saliva protein.
  • Such a medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • the medium may comprise complex nutrients or may be a defined minimal medium.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, which include, but are not limited to, batch, fed-batch, cell recycle, and continuous fermentors. Culturing can also be conducted in shake flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and oxygen content appropriate for the recombinant cell. Such culturing conditions are well withm the expertise of one of ordinary skill m the art.
  • resultant ectoparasite saliva proteins may either remain with the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. col i ; or be retained on the outer surface of a cell or viral membrane.
  • the phrase "recovering the protein” refers simply to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification.
  • Ectoparasite saliva proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, chromatofocusmg and differential solubilization.
  • Ectoparasite saliva proteins are preferably retrieved in "substantially pure” form.
  • substantially pure refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic. For example, an animal being administered dosages of ectoparasite saliva protein isolated from a recombinant cell of the present invention should exhibit no substantial toxicity from contaminants mixed with the protein.
  • Ectoparasite saliva that is substantially free of contaminating material can be collected using a saliva collection apparatus of the present invention (disclosed in related PCT Patent Publication No. WO 96/11,271, published April 18, 1996, by Frank et al.; this publication is incorporated by reference herein in its entirety) .
  • the interior diameter of a preferred chamber of the present invention is preferably about 7.5 cm.
  • the size of a collection means of the present invention is preferably larger than the open end of the 7.5 cm chamber, the size of tne collection means is more preferably about 8 cm.
  • ectoparasite saliva products can be extracted from a collection means (described related PCT Patent Publication No.
  • WO 96/11,271 by contacting a collection means with a Tris buffer containing sodium chloride, alcohol and Tris.
  • a more preferred extraction buffer includes 2.5 M NaCl, 5? IPA and 20 mM Tris, about pH 8.0 to about pH 8.3. Suitable extraction times for elutmg proteins and other products from the collection means using the Tris buffer are described in detail in the Examples.
  • HIC resins include any resins that bind protein at high salt concentrations.
  • Preferred HIC resins include, for example, butyl-, octyl- and phenyl-substrate conjugated resins.
  • a more preferred resm includes a phenyl-sepharose resm.
  • extracted flea saliva proteins contained m a Tr s buffer of the present invention can be contacted with a HIC resm to bind the flea saliva proteins to the resin.
  • a “mimetope” refers to any compound that is able to mimic the ability of an isolated ectoparasite saliva protein of the present invention to carry out its function (e.g., anti- coagulation, anti-complement, vasodialators, proteases, acid phosphatases or detecting and/or treating the hypersensitivity of an animal susceptible to or having allergic dermatitis) .
  • a mimetope can be a peptide that has been modified to decrease its susceptibility to degradation but that still retains the desired activity.
  • mimetopes include, but are not limited to, carbohydrate- based compounds, lipid-based compounds, nucleic acid-based compounds, natural organic compounds, synthetically derived organic compounds, anti-idiotypic antibodies and/or catalytic antibodies, or fragments thereof.
  • Mimetopes of the present invention can also include non-proteinaceous portions of ectoparasite saliva products having allergenic and/or antigenic activity (e.g., carbohydrate moieties associated with ectoparasite saliva proteins) .
  • a mimetope can be obtained by, for example, screening libraries of synthetic compounds for compounds capable of altering the ability of ectoparasites to feed, or of detecting and/or treating allergic dermatitis resulting from the bites of ectoparasites.
  • a mimetope can also be obtained by, for example, rational drug design.
  • the three-dimensional structure of a compound of the present invention can be analyzed by, for example, nuclear magnetic resonance (NMR) or x-ray crystallography.
  • the three-dimensional structure can then be used to predict structures of potential mimetopes by, for example, computer modeling.
  • the predicted mimetope structures can then be produced by, for example, chemical synthesis, recombinant DNA technology, or by isolating a mimetope from a natural source (e.g., plants, animals, bacteria and fungi) .
  • One embodiment of the present invention is an m vi vo test that is capable of detecting whether an animal is hypersensitive to ectoparasite saliva products.
  • An m vi vo test of the present invention can initially be used to determine if an animal is hypersensitive to ectoparasite saliva products and then used to determine if an animal is hypersensitive to a particular ectoparasite saliva component, in particular to an ectoparasite saliva protein.
  • An i n vi vo hypersensitivity test of the present invention is particularly useful for identifying animals susceptible to or having allergic dermatitis.
  • An m vi vo hypersensitivity test of the present invention is even more useful for identifying animals susceptible to or having FAD.
  • a suitable in vi vo hypersensitivity test of the present invention can be, but is not limited to, a skin test comprising administering (e.g., mtradermally injecting or superficial scratching) an effective amount of a formulation containing at least one ectoparasite saliva product, or a mimetope thereof.
  • administering e.g., mtradermally injecting or superficial scratching
  • a formulation containing at least one ectoparasite saliva product, or a mimetope thereof e.g., mtradermally injecting or superficial scratching
  • Methods to conduct sk tests of the present invention are known to those of skill in the art and are briefly disclosed herein.
  • Suitable formulations to use in an m vi vo skin test include one or more isolated ectoparasite saliva proteins of the present invention.
  • a suitable amount of ectoparasite saliva protein for use a skin test of the present invention can vary widely depending on the allergenicity of the product used m the test and on the site at which the product is delivered.
  • Suitable amounts of ectoparasite saliva proteins for use in a skin test of the present invention include an amount capable of forming reaction, such as a detectable wheal or induration (hardness) resulting from an allergic reaction to the product.
  • Preferred amounts of ectoparasite saliva proteins for use m a skin test of the present invention range from about 1 nanogra (ng) to about 500 micrograms ( ⁇ g) , more preferably from about 5 ng to about 300 ⁇ g, and even more preferably from about 10 ng to about 50 ⁇ g of ectoparasite saliva proteins. It is to be appreciated by those of skill m the art that such amounts will vary depending upon the allergenicity of the protein (s) being administered.
  • ectoparasite saliva proteins of the present invention can be combined with an immunopotentiator (e.g., carriers or adjuvants of the present invention as defined in detail below) .
  • an immunopotentiator e.g., carriers or adjuvants of the present invention as defined in detail below.
  • a novel aspect, however, of the present invention is that an ectoparasite saliva protein of the present invention can induce a hypersensitive response m the absence of an lmmunopotentiator.
  • a skin test of the present invention further comprises administering a control solution to an animal.
  • a control solution can include a negative control solution and/or a positive control solution.
  • a positive control solution of the present invention contains an effective amount of at least one compound known to induce a hypersensitive response when administered to an animal.
  • a preferred compound for use as positive control solution includes, but is not limited to, histamine.
  • a negative control solution of the present invention can comprise a solution that is known not to induce a hypersensitive response when administered to an animal.
  • a negative control solution can comprise a solution having compounds essentially incapable of inducing a hypersensitive response or simply a buffer used to prepare the formulation, such as saline.
  • An example of a preferred negative control solution is phenolated phosphate buffered saline (available from Greer Laboratories, Inc., Lenoir, NC) .
  • Hypersensitivity of an animal to one or more formulations of the present invention can be evaluated by measuring reactions (e.g., wheal size, induration or hardness; using techniques known to those skilled m the art) resulting from administration of one or more experimental sample (s) and control sample (s) into an animal and comparing the reactions to the experimental sample (s) with reactions resulting from administration of one or more control solution.
  • Preferred devices for mtradermal injections include individual syringes.
  • Preferred devices for scratching include devices that permit the administration of a number of samples at one time.
  • the hypersensitivity of an animal can be evaluated by determining if the reaction resulting from administration of a formulation of the present invention is larger than the reaction resulting from administration of a negative control, and/or by determining if the reaction resulting from administration of the formulation is at least about the same size as the reaction resulting from administration of a positive control solution.
  • an experimental sample produces a reaction greater than or equal to the size of a wheal produced by administration of a positive control sample to an animal, then that animal is hypersensitive to the experimental sample.
  • an experimental sample produces a reaction similar to the reaction produced by administration of a negative control sample to an animal, then that animal is not hypersensitive to the experimental sample.
  • Preferred wheal sizes for evaluation of the hypersensitivity of an animal range from about 16 mm to about 8 mm, more preferably from about 15 mm to about 9 mm, and even more preferably from about 14 mm to about 10 mm in diameter.
  • the ability or inability of an animal to exhibit an immediate hypersensitive response to a formulation of the present invention is determined by measuring wheal sizes from about 2 minutes to about 30 minutes after administration of a sample, more preferably from about 10 minutes to about 25 minutes after administration of a sample, and even more preferably about 15 minutes after administration of a sample.
  • the ability or inability of an animal to exhibit a delayed hypersensitive response to a formulation of the present invention is determined by measuring induration and/or erythema from about 18 hours to about 30 hours after administration of a sample, more preferably from about 20 hours to about 28 hours after administration of a sample, and even more preferably at about 24 hours after administration of a sample.
  • a delayed hypersensitivity response can also be measured using other techniques such as by determining, using techniques known to those of skill in the art, the extent of cell infiltrate at the site of administration during the time periods defined directly above.
  • a skin test of the present invention comprises mtradermally injecting into an animal at a given site an effective amount of a formulation that includes at least one flea saliva protein of the present invention, and mtradermally injecting an effective amount of a control solution into the same animal at a different site.
  • a formulation that includes at least one flea saliva protein of the present invention
  • mtradermally injecting an effective amount of a control solution into the same animal at a different site It is withm the scope of one of skill the art to use devices capable of delivering multiple samples simultaneously at a number of sites, preferably enabling concurrent evaluation of numerous formulations.
  • One preferred formulation comprises flea saliva products collected in accordance with the present invention. Also preferred are formulations comprising one or more recombmantly produced flea saliva proteins.
  • Suitable flea saliva proteins for use with a skin test of the present invention include proteins having an ammo acid sequence such as is listed m the Sequence Listing herein, or homologues thereof.
  • a preferred positive control sample can be a sample comprising histam e.
  • a preferred negative control sample can be a sample comprising diluent.
  • Animals suitable and preferred to test for hypersensitivity to ectoparasite saliva proteins using a skin test of the present invention are disclosed herein. Particularly preferred animals to test with a skin test of the present invention include dogs, cats and horses, with dogs and cats being even more preferred.
  • Another embodiment of the present invention is an m vi tro immunoabsorbent test that is capable of detecting the presence of an antibody capable of binding to one or more ectoparasite saliva proteins of the present invention by contacting a putative antibody-contammg solution with a solution containing ectoparasite saliva proteins m such a manner that lmmunocomplexes can form and be detected.
  • an in vi tro immunoabsorbent test of the present invention is particularly useful for identifying animals susceptible to or having allergic dermatitis by demonstrating that an animal has been previously exposed to an ectoparasite saliva antigen and, therefore may be hypersensitive to further exposure to an ectoparasite saliva antigen.
  • an m vi tro hypersensitivity test of the present invention can be, but is not limited to, an immunoabsorbent test comprising: (a) contacting a formulation of the present invention with a body fluid from an animal under conditions sufficient for formation of an lmmunocomplex between the formulation and antibodies, if present, in the body fluid; and (b) determining the amount of lmmunocomplex formed, wherem formation of the lmmunocomplex indicates that the animal is susceptible to or has allergic dermatitis.
  • the immunoabsorbent test is particularly useful for the detection of IgE antibodies m the body fluid, thereby indicating immediate hypersensitivity in the animal. Determining the amount of lmmunocomplex formed can include the step of separating depending on the mode of detection. Immunoabsorbent assays can be a variety of protocols and can be set-up by those of skill in the art.
  • a preferred immunoabsorbent test of the present invention comprises a first step of coating one or more portions of a solid substrate with a suitable amount of one or more ectoparasite saliva proteins of the present invention or a mimetope thereof, and of coating one or more other portions of the (or another) solid substrate with a suitable amount of positive and/or negative control solutions of the present invention.
  • a preferred solid substrate of the present invention can include, but is not limited to, an ELISA plate, a dipstick, a radioimmunoassay plate, agarose beads, plastic beads, immunoblot membranes and paper; a more preferred solid substrate includes an ELISA plate, a dipstick or a radioimmunoassay plate, with an ELISA plate and a dipstick being even more preferred.
  • a dipstick refers to any solid material having a surface to which antibodies can be bound, such solid material havmg a stick-like shape capable if being inserted into a test tube. Suitable and preferred flea saliva proteins for use with an m vi tro hypersensitivity test of the present invention are as disclosed for a skin test of the present invention.
  • a second step of a preferred m vi tro hypersensitivity test of the present invention comprises contacting the coated substrate with a body fluid, such as serum, plasma or whole blood, from an animal susceptible to allergic dermatitis in such a manner as to allow antibodies contained in the body fluid that are capable of binding to ectoparasite saliva products to bind to such products bound to the substrate to form immunocomplexes. Excess body fluid and antibodies are then washed from the substrate.
  • the body fluid can be pretreated to remove at least some of the other isotypes of immunoglobulin and/or other proteins, such as albumin, present in the fluid.
  • Such removal can include, but is not limited to, contacting the body fluid with a material, such a Protein G, to remove IgG antibodies and/or affinity purifying the IgE antibodies from other components of the body fluid by exposing the fluid to, for example, Concanavalm A (Con-A) .
  • a third step of a preferred m vi tro hypersensitivity test of the present invention comprises contacting the immunocomplexes bound to the substrate with a compound capable of binding to the immunocomplexes, such as a secondary antibody or other compound that is capable of binding to the heavy chain of allergy-related antibodies produced by animals allergic to ectoparasites, such a manner that the compound (s) can bind to the immunocomplexes.
  • Preferred binding compounds include, but are not limited to, secondary antibodies capable of binding to the heavy chain of IgE antibodies and Fc receptors (FcR) that bind to IgE antibodies (i.e., epsilon FcR), including single chains of an FcR (e.g., the alpha chain of an epsilon FcR) , as well as truncated forms with or without transmembrane domains.
  • FcR Fc receptors
  • Such labels include, but are not limited to, a radioactive label, an enzyme capable of producing a color reaction upon contact with a substrate, a fluorescent label, a chemiluminescent label, a chromophoric label or a compound capable of being bound by another compound.
  • Preferred labels include, but are not limited to, fluorescein, radioisotopes, alkaline phosphatases, biotin, avidin, or peroxidases .
  • a fourth step of a preferred m vi tro hypersensitivity test of the present invention comprises measuring the amount of detectable label bound to the solid substrate using techniques known to those of skill in the art. It is withm the scope of the present invention that the amount of antibody from the body fluid bound to the substrate can be determined using one or more layers of secondary antibodies or other binding compounds. For example, an untagged secondary antibody can be bound to a serum antibody and the untagged secondary antibody can then be bound by a tagged tertiary antibody.
  • a hypersensitive animal is identified by comparing the level of lmmunocomplex formation using samples of body fluid with the level of lmmunocomplex formation using control samples.
  • An lmmunocomplex refers to a complex comprising an antibody and its ligand (i.e., antigen) .
  • immunocomplexes form using positive control samples and do not form using negative control samples.
  • a body fluid sample results in lmmunocomplex formation greater than or equal to lmmunocomplex formation using a positive control sample, then the animal from which the fluid was taken is hypersensitive to the ectoparasite saliva product bound to the substrate.
  • a preferred embodiment of an in vi tro hypersensitivity test of the present invention comprises the steps of: (a) contacting an ELISA plate, which is coated with a suitable amount of flea saliva extract (disclosed in related PCT Patent Publication No. WO 96/11,271, published April 18, 1996, by Frank et al . ; this publication is incorporated by reference herein its entirety), including FS-1, FS-2, FS-3 and/or one or more flea saliva proteins (disclosed in related PCT Patent Publication No.
  • step (b) identifying whether immunocomplexes are formed by step (a) by assaying for the presence of such immunocomplexes by (i) contacting the plate with an antibody that specifically binds to IgE or other compounds capable of binding to such immunocomplexes, such as an epsilon Fc receptor, and (ii) determining whether such an antibody or other compound is bound thereto.
  • kits useful for identification of an animal susceptible to or having allergic dermatitis As used herein, a suspect animal is an animal to be tested.
  • a kit of the present invention comprises a formulation of the present invention and a means for determining if an animal is susceptible to or has allergic dermatitis, in which the formulation is used to identify animals susceptible to or having allergic dermatitis.
  • a means for determining if an animal is susceptible to or has allergic dermatitis can include an in vi vo or in vi tro hypersensitivity test of the present invention as described in detail above.
  • a kit of the present invention further comprises at least one control solution such as those disclosed herein.
  • a preferred kit of the present invention comprises the elements useful for performing an immunoassay.
  • a kit of the present invention can comprise one or more experimental samples ⁇ i.e., formulations of the present invention) and one or more control samples bound to at least one pre- packed dipstick or ELISA plate, and the necessary means for detecting immunocomplex formation (e.g., labeled secondary antibodies or other binding compounds and any necessary solutions needed to resolve such labels, as described in detail above) between antibodies contained in the bodily fluid of the animal being tested and the proteins bound to the dipstick or ELISA plate. It is within the scope of the invention that the kit can comprise simply a formulation of the present invention and that the detecting means can be provided in another way.
  • An alternative preferred kit of the present invention comprises elements useful for performing a skin test.
  • a kit of the present invention can comprise at least one pre ⁇ packed syringe and needle apparatus containing one or more experimental samples and/or one or more control samples.
  • two or more different m vi vo and/or in vi tro tests can be used m combination for diagnostic purposes.
  • the immediate hypersensitivity of an animal to an ectoparasite saliva allergen can be tested using an m vi tro immunoabsorbent test capable of detecting IgE antibodies specific for an ectoparasite saliva allergen m the animal's bodily fluid. While most animals that display delayed hypersensitivity to an ectoparasite saliva allergen also display immediate hypersensitivity to the allergen, a small number of animals that display delayed hypersensitivity to an allergen do not display immediate hypersensitivity to the allergen. In such cases, following negative results from the IgE-specific m vi tro test, the delayed hypersensitivity of the animal to an ectoparasite saliva allergen can be tested using an in vi vo test of the present invention.
  • Another aspect of the present invention includes treating animals susceptible to or havmg allergic dermatitis, with a formulation of the present invention.
  • the term treatment can refer to the regulation of a hypersensitive response by an animal to bites from ectoparasites. Regulation can include, for example, lmmunomodulation of cells involved in the animal's hypersensitive response or alteration of the ability of an ectoparasite to introduce allergens into an animal, for example by inhibiting the anti-coagulation activity of a saliva enzyme, thereby impairing the ability of the arthropod to penetrate the dermis of an animal and feed. lmmunomodulation can include modulating the activity of molecules typically involved in an immune response
  • lmmunomodulation refers to modulation of antigen: antibody interactions resulting m inflammatory responses, immunosuppression, and immunotolerization of cells involved in a hypersensitive response.
  • Immunosuppression refers to inhibiting an immune response by, for example, killing particular cells involved in the immune response.
  • Immunotolerization refers to inhibiting an immune response by anergiz g (i.e., diminishing reactivity of a T cell to an antigen) particular cells involved in the immune response. Suitable and preferred ectoparasites against which to treat an animal are disclosed herein.
  • a particularly preferred formulation of the present invention is used to treat FAD.
  • One embodiment of the present invention is a therapeutic composition that, when administered to an animal in an effective manner, is useful for lmmunomodulating the immune response of the animal (i.e., lmmunomodulating the animal) so as to block (i.e., to inhibit, reduce or substantially prevent) a hypersensitive response by the animal upon subsequent exposure to allergenic components transmitted through bites from ectoparasites.
  • Such a therapeutic composition is useful for lmmunomodulating animals known to be hypersensitive to ectoparasite saliva products and animals susceptible to hypersensitive responses against ectoparasite saliva products .
  • One embodiment of the present invention is a therapeutic composition that includes de-sensitizmg compounds capable of inhibiting an immune response to an ectoparasite saliva protein of the present invention.
  • de-sensitizmg compounds include blocking compounds, toleragens and/or suppressor compounds.
  • Blocking compounds comprise compounds capable of modulating antigen:antibody interactions that can result in inflammatory responses
  • toleragens are compounds capable of lmmunotole ⁇ zmg an animal
  • suppressor compounds are capable of lmmunosuppressmg an animal.
  • a de-sensitizmg compound of the present invention can be soluble or membrane-bound.
  • Membrane-bound de-sensitizmg compounds can be associated with biomembranes, including cells, liposomes, planar membranes, cochleates or micelles.
  • a soluble de ⁇ sensitizing compound of the present invention is useful for: (1) inhibiting a Type I hypersensitivity reaction by blocking IgE:ant ⁇ gen mediated de-granulation of mast cells; (2) inhibiting a Type III hypersensitivity reaction by blocking IgG:antigen complex formation leading to complement destruction of cells; and (3) inhibiting a Type IV hypersensitivity reaction by blocking T helper cell stimulation of cytokme secretion by macrophages.
  • a membrane-bound de-sensitizmg compound of the present invention is useful for: (1) inhibiting a Type II hypersensitivity reaction by blocking IgG:antigen complex formation on the surface of cells leading to complement destruction of cells; (2) inhibiting a Type II hypersensitivity reaction by blocking IgG regulated signal transduction in immune cells; and (3) inhibiting a Type IV hypersensitivity reaction • by blocking T cytotoxic cell killing of antigen-bearing cells.
  • a de-sensitiz g compound of the present invention can also be covalently linked to a ligand molecule capable of targeting the de-sensitizmg compound to a specific cell involved in a hypersensitive response to ectoparasite saliva products.
  • Appropriate ligands with which to link a de-sensitizmg compound include, for example, at least a portion of an immunoglobulin molecule, cytokmes, lectins, heterologous allergens, CD8 molecules, CD4 molecules or major histocompatibility molecules (e.g., MHC class I or MHC class II molecules) .
  • Preferred portions of immunoglobulin molecules to link to a de-sensitizmg compound include variable regions capable of binding to immune cell specific surface molecules and constant regions capable of binding to Fc receptors on immune cells, m particular IgE constant regions.
  • Preferred CD8 molecules include at least the extracellular functional domains of the ⁇ chain of CD8.
  • Preferred CD4 molecules include at least the extracellular functional domains of CD4.
  • An immune cell refers to a cell involved in an immune response, m particular, cells having MHC class I or MHC class II molecules.
  • Preferred immune cells include antigen presenting cells, T cells and B cells.
  • a therapeutic composition of the present invention includes ectoparasite saliva products of the present invention, or mimetopes thereof.
  • Preferred therapeutic compositions include formulations comprising ectoparasite saliva extracts or at least one ectoparasite saliva product (preferably protein) of the present invention or mimetopes thereof.
  • Suitable therapeutic compositions of the present invention for treating flea allergy dermatitis include flea saliva extracts (such as those disclosed m related PCT Patent Publication No. WO 96/11,271) and other formulations including at least one flea saliva protein, or a mimetope thereof.
  • Preferred therapeutic compositions include FS-1, FS-2 and/or FS-3 (such as those disclosed related PCT Patent Publication No. WO 96/11,271) as well as at least a portion of at least one flea saliva protein that can be isolated from FS-1, FS-2 and/or FS-3.
  • preferred formulations for use as therapeutic compositions include FS-1, FS-2, FS-3, and/or at least a portion of one or more of the proteins havmg an ammo acid sequence including SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO:72, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO: 78 and SEQ ID NO: 87.
  • a therapeutic composition can include ectoparasite products of the present invention associated with a suitable excipient.
  • a therapeutic composition of the present invention can be formulated m an excipient that the animal to be treated can tolerate.
  • Preferred excipients are capable of maintaining a product of the present invention a form that is capable of being bound by cells involved in an allergic response in an animal such that the cells are stimulated to initiate or enhance an immune response. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.
  • Nonaqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or tnglycerides may also be used.
  • compositions include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran.
  • Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.
  • buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol.
  • Standard formulations can either be liquid mjectables or solids which can be taken up m a suitable liquid as a suspension or solution for injection.
  • the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
  • a therapeutic composition of the present invention can also comprise a carrier or adjuvant, although it is to be appreciated that an advantage of saliva products of the present invention is that adjuvants and/or carriers are not required for administration.
  • Adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen.
  • Suitable adjuvants include, but are not limited to, cytokmes, chemokmes, and compounds that induce the production of cytokmes and chemokmes (e.g., granulocyte macrophage colony stimulating factor [GM-CSF] , macrophage colony stimulating factor [M-CSF] , granulocyte colony stimulating factor [G-CSF] , colony stimulating factor [CSF], erythropoietm [EPO], mterleukm-2 [IL-2], mterleuk ⁇ n-3 [IL-3], mterleukm-5 [IL-5], mterleukm-6 [IL-6], mterleukm-7 [IL-7], mterleukm-8 [IL-8], mterleukm-10 [IL-10], mterleukm-12 [IL-12], gamma mterferon [IFN- ⁇ ], mterferon gamma inducing factor [IGIF], transforming growth factor beta, RANTES [regulated upon activation, normal
  • Protein adjuvants of the present invention can be delivered m the form of the protein themselves or of nucleic acid molecules encoding such proteins using the methods described herem.
  • Carriers are typically compounds that increase the half-life of a therapeutic composition m the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, bacteria, viruses, oils, esters, ana glycols .
  • One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a therapeutic composition of the present invention into the bloodstream of an animal.
  • Suitable controlled release formulations include, but are not limited to, biocompatible (including biodegradable) polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems.
  • Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel m si t u .
  • the present invention also includes a recombinant virus particle therapeutic composition.
  • a composition includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed an animal after administration.
  • the recombinant molecule is packaging-deficient.
  • a number of recombinant virus particles can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses .
  • Preferred recombinant particle viruses are those based on alphaviruses (such as Sindbis virus) , herpesviruses and poxviruses.
  • a recombinant virus particle therapeutic composition of the present invention When administered to an animal, infects cells withm the immunized animal and directs the production of a protective protein or RNA nucleic acid molecule that is capable of protecting the animal from allergic dermatitis caused by the bites of ectoparasites.
  • a recombinant virus particle comprising a nucleic acid molecule encoding one or more ectoparasite saliva protein of the present invention is administered according to a protocol that results m the tolerization of an animal against ectoparasite saliva allergens.
  • a nucleic acid molecule of the present invention can be delivered to an animal as a naked (i.e., not packaged m a viral coat or cellular membrane) nucleic acid vaccine (e.g., as naked DNA or RNA molecules, such as is taught, for example m Wolff et al . , 1990, Sci ence 247, 1465-1468) .
  • a naked nucleic acid vaccine of the present invention includes a nucleic acid molecule of the present invention and preferably includes a recombinant molecule of the present invention that preferably is replication, or otherwise amplification, competent.
  • a naked nucleic acid vaccine of the present invention can comprise one or more nucleic acid molecules of the present invention m the form of, for example, a dicistromc recombinant molecule.
  • Preferred naked nucleic acid vaccines include at least a portion of a viral genome (i.e., a viral vector) .
  • Preferred viral vectors include those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses, with those based on alphaviruses (such as Smdbis or Semliki virus), species- specific herpesviruses and species-specific poxviruses Demg particularly preferred. Any suitable transcription control sequence can be used, including those disclosed as suitable for protein production.
  • transcription control sequence include cytomegalovirus intermediate early (preferably in conjunction with Intron- A) , Rous Sarcoma Virus long terminal repeat, and tissue-specific transcription control sequences, as well as transcription control sequences endogenous to viral vectors if viral vectors are used.
  • the incorporation of "strong" poly (A) sequences are also preferred.
  • Naked nucleic acid vaccines of the present invention can be administered in a variety of ways, with intramuscular, subcutaneous, mtradermal, transdermal, tranasal and oral routes of administration being preferred.
  • An example of one embodiment is disclosed in PCT Patent Publication No. WO 95/05853, published March 2, 1995.
  • a preferred single dose of a naked nucleic acid vaccine ranges from about 1 nanogram (ng) to about 100 ⁇ g, depending on the route of administration and/or method of delivery, as can be determined by those skilled the art. Suitable delivery methods include, for example, by injection, as drops, aerosolized, oral and/or topical.
  • Naked DNA of the present invention can be contained m an aqueous excipient (e.g., phosphate buffered saline) alone or a carrier (e.g., lipid-based vehicles) .
  • compositions of the present invention can be sterilized by conventional methods which do not result in protein degradation (e.g., filtration) and/or lyophilized.
  • a therapeutic composition of the present invention can be administered to any animal susceptible to ectoparasite infestation as herein described.
  • Acceptable protocols by which to administer therapeutic compositions of the present invention m an effective manner can vary according to individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art.
  • An effective dose refers to a dose capable of treating an animal against hypersensitivity to ectoparasite saliva allergens. Effective doses can vary depending upon, for example, the therapeutic composition used, the arthropod from which the composition was derived, and the size and type of the recipient animal.
  • Effective doses to immunomodulate an animal against ectoparasite saliva allergens include doses administered over time that are capable of alleviating a hypersensitive response by an animal to ectoparasite saliva allergens.
  • a first tolerizing dose can comprise an amount of a therapeutic composition of the present invention that causes a minimal hypersensitive response when administered to a hypersensitive animal.
  • a second tolerizing dose can comprise a greater amount of the same therapeutic composition than the first dose.
  • Effective tolerizing doses can comprise increasing concentrations of the therapeutic composition necessary to tolerize an animal such that the animal does not have a hypersensitive response to the bite of an ectoparasite.
  • An effective dose to desensitize an animal can comprise a concentration of a therapeutic composition of the present invention sufficient to block an animal from havmg a hypersensitive response to the bite of an ectoparasite.
  • Effective desensitizing doses can include repeated doses having concentrations of a therapeutic composition that cause a minimal hypersensitive response when administered to a hypersensitive animal.
  • a suitable single dose is a dose that is capable of treating an animal against hypersensitivity to ectoparasite saliva allergens when administered one or more times over a suitable time period.
  • a preferred single dose of an ectoparasite saliva product, or mimetope therapeutic composition is from about 0.5 ng to about 1 g of the therapeutic composition per kilogram body weight of the animal.
  • Further treatments with the therapeutic composition can be administered from about 1 hour to 1 year after the original administration. Further treatments with the therapeutic composition preferably are administered when the animal is no longer protected from hypersensitive responses to ectoparasite.
  • Particular administration doses and schedules can be developed by one of skill in the art based upon the parameters discussed above. Modes of administration can include, but are not limited to, subcutaneous, mtradermal, intravenous, nasal, oral, transdermal and intramuscular routes.
  • a therapeutic composition of the present invention can be used in conjunction with other compounds capable of modifying an animal's hypersensitivity to ectoparasite bites.
  • an animal can be treated with compounds capable of modifying the function of a cell involved in a hypersensitive response, compounds that reduce allergic reactions, such as by systemic agents or anti-mflammatory agents (e.g., anti-histammes, anti-steroid reagents, anti- inflammatory reagents and reagents that drive immunoglobulin heavy chain class switching from IgE to IgG) .
  • systemic agents or anti-mflammatory agents e.g., anti-histammes, anti-steroid reagents, anti- inflammatory reagents and reagents that drive immunoglobulin heavy chain class switching from IgE to IgG.
  • Suitable compounds useful for modifying the function of a cell involved in a hypersensitive response include, but are not limited to, antlhistamines, cromolyn sodium, theophyllme, cyclosporm A, adrenalin, cortisone, compounds capable of regulating cellular signal transduction, compounds capable of regulating adenosme 3',5'-cyclic phosphate (cAMP) activity, and compounds that block IgE activity, such as peptides from IgE or IgE specific Fc receptors, antibodies specific for peptides from IgE or IgE-specific Fc receptors, or antibodies capable of blocking binding of IgE to Fc receptors.
  • antlhistamines cromolyn sodium, theophyllme, cyclosporm A, adrenalin, cortisone
  • compounds capable of regulating cellular signal transduction compounds capable of regulating adenosme 3',5'-cyclic phosphate (cAMP) activity
  • cAMP adeno
  • a preferred method for prescribing treatment for flea allergy dermatitis comprises: (1) mtradermally injecting nto an animal at one site an effective amount of a formulation containing at least one flea saliva antigen of the present invention, or a mimetope thereof (suitable and preferred formulations are disclosed herein) ; (2) mtradermally injecting into the animal at a second site an effective amount of a control solution; (3) evaluating if the animal has flea allergy dermatitis by measuring and comparing the wheal size resulting from injection of the formulation with the wheal size resulting from injection of the control solution; and (4) prescribing a treatment for the flea allergy dermatitis.
  • An alternative preferred method for prescribing treatment for flea allergy dermatitis comprises: (1) contacting a first portion of a sample of bodily fluid obtained from an animal to be tested with an effective amount of a formulation containing at least one flea saliva antigen, or a mimetope thereof (suitable and preferred formulations are disclosed herein) to form a first lmmunocomplex solution; (2) contacting a positive control antibody to form a second lmmunocomplex solution; (3) evaluating if the animal has flea allergy dermatitis by measuring and comparing the amount of lmmunocomplex formation in the first and second lmmunocomplex solutions; and (4) prescribing a treatment for the flea allergy dermatitis.
  • Another aspect of the present invention includes a method for monitoring animals susceptible to or havmg allergic dermatitis, using a formulation of the present invention. In vi vo and in vi tro tests of the present invention can be used to test animals for allergic dermatitis prior to and following any treatment for allergic dermatitis.
  • a preferred method to monitor treatment of flea allergy dermatitis comprises: (1) tradermally injecting an animal at one site with an effective amount of a formulation containing at least one flea saliva protein, or a mimetope thereof (suitable and preferred formulations are disclosed herein); (2) mtradermally injecting an effective amount of a control solution into the animal at a second site; and (3) determining if the animal is desensitized to flea saliva antigens by measuring and comparing the wheal size resulting from injection of the formulation with the wheal size resulting from injection of the control solution.
  • An alternative preferred method to monitor treatment of flea allergy dermatitis comprises: (1) contacting a first portion of a sample of bodily fluid obtained from an animal to be tested with an effective amount of a formulation containing at least one flea saliva protein or mimetope thereof (suitable and preferred formulations are disclosed herein) to form a first lmmunocomplex solution; (2) contacting a positive control antibody to form a second lmmunocomplex solution; and (3) determining if the animal is desensitized to flea saliva antigens by measuring and comparing the amount of lmmunocomplex formation m the first and second lmmunocomplex solutions.
  • the present invention also includes antibodies capable of selectively binding to an ectoparasite saliva protein, or mimetope thereof.
  • an antibody is herein referred to as an anti-ectoparasite saliva protein antibody.
  • selectively binds to refers to the ability of such an antibody to preferentially bind to ectoparasite saliva proteins and mimetopes thereof.
  • the present invention includes antibodies capable of selectively binding to flea saliva proteins.
  • Binding can be measured using a variety of methods known to those skilled m the art including immunoblot assays, immunoprecipitation assays, enzyme immunoassays (e.g., ELISA) , radioimmunoassays, immunofluorescent antibody assays and lmmunoelectron microscopy; see, for example, Sambrook et al . , ibid.
  • Antibodies of the present invention can be either polyclonal or monoclonal antibodies.
  • Antibodies of the present invention include functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protein or mimetope used to obtain the antibodies.
  • an antibody of the present invention has a single site binding affinity of from about IO 1 M ⁇ to about IO 1 - M ⁇ * for a flea saliva product of the present invention.
  • a preferred method to produce antibodies of the present invention includes administering to an animal an effective amount of an ectoparasite saliva protein or mimetope thereof to produce the antibody and recovering the antibodies.
  • Antibodies raised against defined proteins or mimetopes can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference a diagnostic assay or side effects if used in a therapeutic composition.
  • Antibodies of the present invention have a variety of potential uses that are withm the scope of the present invention.
  • such antibodies can be used (a) as vaccines to passively immunize an animal m order to protect the animal from allergic dermatitis, (b) as positive controls in test kits, and/or (c) as tools to recover desired ectoparasite saliva proteins from a mixture of proteins and other contaminants.
  • the following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.
  • This example describes the ammo acid sequence analysis of additional isolated flea saliva proteins from FS-1 extract and eluted from DE-81 filters.
  • FS-1 flea saliva extract and flea saliva product eluted from DE-81 filters were collected using techniques described m Example 2 of related PCT Publication No. WO 96/11,271.
  • standard purification techniques e.g., C4 reverse phase chromatography; SDS-PAGE gel electrophoresis and blotting; and/or flow through electrophoresis
  • peak M contained fspJ, fspL and fspN proteins
  • fspM(G), fspM(H), fspM(I), fspM(J), fspM(K), fspM(L) and fspM(M) Flea saliva protein fspM(G), having a molecular weight of about 37 kD, had an N-termmal partial ammo acid sequence of M R G N H V F L
  • Flea saliva protein fspM(M) was recovered from peak M and subjected to ammo acid sequence analysis as described in Example 4 of related PCT Publication No. WO 96/11,271.
  • Flea saliva protein fsp(M) having a molecular weight of about 31 kD, had an N-terminal partial ammo acid sequence of Y F N D Q I K S V M E P X V F K Y P X A X L, denoted SEQ ID NO:7.
  • a Genbank homology search revealed no significant homology between known ammo acid sequences and those determined for fspM(G), fspM(H), fspM(I), fspM(J), fspM(K), fspM(L) and fspM(M) .
  • Example 2 A Genbank homology search revealed no significant homology between known ammo acid sequences and those determined for fspM(G), fspM(H), fspM(I), fspM(J), fspM(K), fspM(L) and fspM(M) .
  • This example describes the isolation of nucleic acid molecules encoding at least a portion of a fspG flea saliva protein. This example also describes expression of a fspG protein by bacteria.
  • A. Isolation of fspG4 nucleic acid molecules The partial N-termmal ammo acid sequence of fspG2 (i.e., SEQ ID NO: 29 of related PCT Publication No. WO 96/11,271) was used to synthesize degenerate antisense Primer G2-2, having the nucleic acid sequence 5' TGR TTT CCW ATR AAR TCT TC 3 ' , denoted SEQ ID NO: 8.
  • Primer G2-2 was used combination with the M13 reverse primer (SEQ ID NO: 40; described m Example 7 of related PCT Publication No.
  • nfspG4_ is presented as SEQ ID NO: 9.
  • nfspG4 225 was used to synthesize sense Primer G5, havmg nucleic acid sequence 5' AAT TCG GCA CGA GTG 3', denoted SEQ ID NO: 10.
  • Primer G5 was used in combination with the M13 universal primer (SEQ ID NO: 19; described m Example 6 of related PCT Publication No. WO 96/11,271), to PCR amplify, as described above, the 3'-terminal portion of the fspG4 gene from the salivary gland cDNA expression library described above in Example 6A of related PCT Publication No. WO 96/11,271) .
  • nfspG4 61(J was approximately 610-bp when visualized on a 1% agarose gel.
  • the nucleotide sequence of the 610-bp PCR fragment was obtained, 565 nucleotides of which are presented as SEQ ID NO: 11.
  • the nucleic acid molecule containing nucleic acid sequence SEQ ID NO: 11 is referred to herein as nfspG4 bj .
  • nucleic acid molecule nfspG4 t encodes a full-length fspG protein of about 90 ammo acids, referred to herein as PfspG4 90 , assuming an open reading frame having a start codon spanning from about nucleotide 45 through about nucleotide 47 of SEQ ID NO: 11 and a stop codon spanning from about nucleotide 315 through about nucleotide 317 of SEQ ID NO: 11.
  • This open reading frame, excluding the stop codon comprises nucleic acid molecule nfspG4 /G of the present invention, the nucleic acid sequence of which is represented herem by SEQ ID NO: 13.
  • PfspG4 90 is denoted herem as SEQ ID N0:12.
  • Residues 20-42 of SEQ ID NO:12 appear to be identical to SEQ ID NO:29 of related PCT Publication No. WO 96/11,271 (N-termmal partial ammo acid sequence of fspG2), except that residue 37 of SEQ ID NO: 12 is a glutamic acid rather than a lysine.
  • residues 38-57 of SEQ ID NO: 12 appear to be identical to SEQ ID NO:30 of related PCT Publication No. WO 96/11,271 (N-terminal partial ammo acid sequence of fspG3) .
  • SEQ ID NO: 11 suggests that the sequence includes a leader segment of about 19 ammo acids followed by a mature protein.
  • the leader sequence is apparently cleaved to form a mature protein termed PfspG4 71 ⁇ denoted SEQ ID NO: 12.
  • PfspG4 71 has a calculated molecular weight of 7536 daltons and calculated pi of about 9.0.
  • PfspG4 90 has a calculated molecular weight of 9657 daltons and calculated pi of about 9.26.
  • a Genbank homology search revealed no significant homology between SEQ ID NO: 11 or SEQ ID NO: 12 and known nucleic acid sequences or known ammo acid sequences, respectively.
  • nfspG4 An about 216-bp DNA fragment of nfspG4 was PCR amplified from nucleic acid molecule nfspG4, using: Primer
  • Primer G8 an antisense primer having the nucleic acid sequence 5' CCG GAA TTC GGT TAT TCG CAA TAA CAG T 3' (£coRI site m bold), denoted SEQ ID NO: 1
  • nfspG4 216 The PCR product, a fragment of about 216 nucleotides, denoted nfspG4 216 , was digested with BamHI and
  • the recombinant molecule was transformed into E . coli to form recombinant cell E . coli :pH ⁇ s-nfspG4 216 .
  • the recombinant cell was cultured and induced as described m
  • the recombinant fusion protein was detected by immunoblot analysis using the T7 Tag monoclonal antibody as described in Example 11A of related PCT Publication No. WO 96/11,271.
  • This example describes the isolation of nucleic acid sequences encoding at least a portion of flea saliva proteins fsp (A) , fspM(B), fspM(C), fspM(D), fspM(E), and fspM(F) .
  • Immunoscreenmg was performed as described m Example 12 of related PCT Publication No. WO 96/11,271.
  • nfspM (A) 897 A nucleotide sequence for a nfspM nucleic acid molecule named nfspM (A) 897 is denoted as SEQ ID NO: 17. Translation of SEQ ID NO: 17 suggests that nucleic acid molecule nfspM (A) 897 encodes a full-length fspM protein of about 157 ammo acids, referred to herem as PfspM(A) 157 , assuming an open reading frame having a start codon spanning from about nucleotide 97 through about nucleotide 99 of SEQ ID NO: 17 and a stop codon spanning from about nucleotide 568 through about nucleotide 570 of SEQ ID NO: 17.
  • This open reading frame excluding the stop codon, comprises nucleic acid molecule nfspM (A) 47] of the present invention, the nucleic acid sequence of which is represented herein by SEQ ID NO: 19.
  • the ammo acid sequence of PfspM (A) ]5/ is denoted SEQ ID NO: 18.
  • PfspM(A) has a calculated molecular weight of about 18,291.68 daltons and calculated pi of about 10.3.
  • a Genbank homology search revealed no significant homology between SEQ ID NO: 17 or SEQ ID NO: 18 and known nucleic acid or ammo acid sequences, respectively.
  • a nucleotide sequence for another nfspM nucleic acid molecule named nfspM(B) 27Qf is denoted as SEQ ID NO:20.
  • Translation of SEQ ID NO:20 suggests that nucleic acid molecule nfspM(B) 2706 encodes a non-full-length fspM protein of about 900 ammo acids, referred to herein as PfspM(B) 900 , assuming an open reading frame having a start codon spanning from about nucleotide 5 through about nucleotide 7 of SEQ ID NO:20.
  • the ammo acid sequence of PfspM(B) 900 is denoted SEQ ID NO:21.
  • PfspM(B) 900 has a calculated molecular weight of about 104,647 daltons and calculated pi of about 5.8.
  • SEQ ID NO:21 and ROK ammo acid sequences spans from about ammo acid 32 through about amino acid 351 of SEQ ID NO:21 and from about ammo acid 1 through about amino acid 900 of the ROK, there being about 75% identity between the two regions.
  • Comparison of the nucleic acid sequence encoding ammo acids from about 326 through about 1285 of the ROK kmase with the corresponding regions, spanning nucleotides from about 98 through about 1075 of nfspM(B) 270f indicate that those regions are about 71% identical.
  • a flea salivary gland cDNA library (prepared as described m Example 6 of related PCT Publication No. WO 96/11,271) was lmmunoscreened with antiserum collected from a rabbit that was immunized with the proteins in peak Ml of the HPLC separation of flea saliva extract described in
  • Nucleotide sequence for a nfspM nucleic acid molecule named nfspM(C) 414 is denoted as SEQ ID NO:22.
  • Translation of SEQ ID NO:22 suggests that nucleic acid molecule nfspM(C) 414 encodes a non-full-length fspM protein of about 137 ammo acids, referred to herein as PfspM(C) 137 , assuming the first residue spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO:22.
  • the ammo acid sequence of PfspMfC), ⁇ is denoted SEQ ID NO: 23.
  • PfspM(C) has a calculated molecular weight of about 14,452 daltons and calculated pi of about 2.81.
  • a Genbank homology search revealed no significant homology between SEQ ID NO:22 or SEQ ID NO: 23 and known nucleic acid sequences or known ammo acid sequences, respectively.
  • a nucleotide sequence for another nfspM nucleic acid molecule named nfspM(D) 273 is denoted as SEQ ID NO: 24.
  • Translation of SEQ ID NO: 24 suggests that nucleic acid molecule nfspM(D) 2 3 encodes a non-full-length fspM protein of about 90 ammo acids, referred to herein as PfspM(D) 90 , assuming the first residue spans from about nucleotide 3 through about nucleotide 5 of SEQ ID NO: 24.
  • the am o acid sequence of PfspM(D) 90 ⁇ s denoted SEQ ID NO:25.
  • PfspM(D) 90 has a calculated molecular weight of about 9, 503 daltons and calculated pi of about 3.01.
  • SEQ ID NO: 24 and SEQ ID NO:25 appear to be substantially similar to SEQ ID NO:22 and SEQ ID NO:23, respectively, suggesting a family of fspM proteins m flea saliva.
  • a flea salivary gland cDNA library (prepared as described in Example 6 as described of related PCT Publication No. WO 96/11,271) was lmmunoscreened with antiserum collected from a rabbit that was immunized with the proteins in peak M2 of the HPLC separation of flea saliva extract described m
  • Example 3 of related PCT Publication No. WO 96/11,271 i.e., fspM2 proteins
  • Immunoscreenmg was performed as described m Example 12 of related PCT Publication No. WO 96/11,271.
  • nfspM (E) 1704 A nucleotide sequence for another nfspM nucleic acid molecule named nfspM (E) 1704 is denoted as SEQ ID NO:26.
  • Translation of SEQ ID NO:26 suggests that nucleic acid molecule nfspM (E) 1704 encodes a full-length fspM protein of about 461 ammo acids, referred to herem as PfspM(E) 461 , assuming the first residue spans from about nucleotide 24 through about nucleotide 26 of SEQ ID NO: 26 and a stop codon spanning from about nucleotide 1407 through about nucleotide 1409 of SEQ ID NO:26.
  • This open reading frame comprises nucleic acid molecule nfspM(E) ⁇ 383 of the present invention, the nucleic acid sequence of which is represented herein by SEQ ID NO: 28.
  • the ammo acid sequence of PfspM(E) 4bl is denoted SEQ ID NO: 27.
  • PfspM(E) 461 has a calculated molecular weight of about 54,139 daltons and calculated pi of about 7.00.
  • a Genbank homology search revealed no significant homology between SEQ ID NO:26 or SEQ ID NO:27 and known nucleic acid sequences or known ammo acid sequences, respectively.
  • nucleotide sequence for another nfspM nucleic acid molecule named nfspM (F) 17 H is denoted as SEQ ID NO:29.
  • Translation of SEQ ID NO:29 suggests that nucleic acid molecule nfspM(F) 175tj encodes a non-full-length fspM protein of about 586 ammo acids, referred to herein as PfspM(F) 586 , assuming an open reading frame having a start codon spanning from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:29.
  • the ammo acid sequence of PfspM(F) 8 ⁇ is denoted SEQ ID NO: 30.
  • PfspM(F) 586 has a calculated molecular weight of about 66,547 daltons and calculated pi of about 4.80.
  • a Genbank homology search revealed no significant homology between SEQ ID NO:29 or SEQ ID NO: 30 and known nucleic acid sequences or known ammo acid sequences, respectively.
  • This Example demonstrates the expression of a fspM protein in E. Coli cells.
  • Flea saliva protein PHIS-PfspM(D) 90 fusion protein was produced m the following manner.
  • An about 305-bp DNA fragment, referred to herein as nfspM(D) 30 was isolated from nfspM(D) 293 (denoted SEQ ID NO: 31) subcloned into pBluescript plasmid by digesting the nfspM(D) -containing plasmid with BamHI and X ol restriction endonucleases. The digestion product was gel purified and subcloned into expression vector pTrcHisB that had been digested with
  • pHis-nfspM(D) J05 The resultant recombinant molecule, referred to herein as pHis-nfspM(D) J05 , was transformed into E. coli HB101 competent cells
  • coli :pH ⁇ s-nfspM (D) 30 lysates using a T7 tag monoclonal antibody (Novagen, Ine) directed against the fusion portion of the recombinant PHis-nfspM(D) 305 fusion protein identified a protein of the appropriate size, namely an about 15,851 kD protein.
  • Example 5 This example describes the isolation of nucleic acid sequences encoding at least a portion of flea saliva proteins fspN(C), fspN(D), fspN(E), fspN(F), fspN(G), fspN(H), fspN(I), fspN(J), fspN(K), fspN(L), fspN(M), fspN(N) and fspN(O) .
  • Serum was obtained from the artificially sensitized dog CQQ2 (described in Example 8 of related PCT Publication No. WO 96/11,271) . About 10 ml of antiserum was incubated with protein G-Sepharose (5 ml) over night at 4°C. B. Immunoscreenmg with IgE enriched antiserum
  • LB Lu ⁇ a-Bertani
  • the filters were then removed and washed with TBST (20 mM T ⁇ s-HCl pH 7.5, 150 mM NaCl, 0.05 Tween-20) .
  • the filters were blocked with 5% dry milk in TBST for 2 hours at room temperature.
  • Different filters were then incubated first with either IgE enriched CQQ2 antiserum or antiserum obtained from dogs infected with Dirofilari a immi ti s ) at 4°C, overnight, then with a monoclonal anti-canme IgE antibody (D-9; gift from the laboratory of Dr. D.J.
  • the m vi vo excision of the pBluescript phagemid from each positive clone was prepared by using ExAss ⁇ stTM/SOLR ','M system (Stratagene; .
  • the pBluescript plasmid was purified by plasmid midi kit (Qiagen), and denatured with NaOH (0.4 N) at 37 C C for 15 mm.
  • the denatured plasmid was precipitated by ethanol and nucleic acid sequence obtained.
  • a nucleotide sequence for a nfspN nucleic acid molecule named nfspN(C) 335 is denoted as SEQ ID NO: 32.
  • a Genbank homology search revealed some similarity between SEQ ID NO: 32 and ⁇ bosomal protein S6.
  • nfspN(D) 396 A nucleotide sequence for another nfspN nucleic acid molecule named nfspN(D) 396 is denoted as SEQ ID NO: 33.
  • a Genbank homology search revealed some similarity between SEQ ID NO: 33 and erythropoiet .
  • nfspN(E) 28 A nucleotide sequence for another nfspN nucleic acid molecule named nfspN(E) 28 , is denoted as SEQ ID NO: 34.
  • Genbank homology search revealed some similarity between
  • SEQ ID NO: 34 glutamic acid-rich protein or heat-shock protein, HSP81.
  • nfspN(F) 22B A nucleotide sequence for another nfspN nucleic acid molecule named nfspN(F) 22B is denoted as SEQ ID NO:35. Nucleic acid sequence for portions of another nfspN nucleic acid molecule, denoted herem as nfspN(G), were obtained. The nucleic acid molecule representing a 5' portion of nfspN(G) named nfspN(G) , ( ⁇ is denoted as SEQ ID NO: 36.
  • nucleic acid molecule nfspN (G) ⁇ 39 encodes a non-full-length fspN(G) protein of about 113 ammo acids, referred to herein as PfspN(G) n3 , assuming the first residue spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 36.
  • the ammo acid sequence of PfspN(G) 113 is denoted SEQ ID NO:37.
  • the nucleic acid molecule representing a 3' portion of nfspN(G) named nfspN(G) 493 is denoted as SEQ ID NO: 38.
  • nucleic acid molecule nfspN(G) 493 encodes a non-full-length fspN(G) protein of about 130 ammo acids, referred to herem as PfspN(G) 130 , assuming the first residue spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 38 and a stop codon spanning from about nucleotide 391 through about nucleotide 393 of SEQ ID NO: 38.
  • the ammo acid sequence of Pfsp (G) o is denoted SEQ ID NO:39.
  • a Genbank homology search revealed some similarity between SEQ ID NO: 36 and SEQ ID NO: 38 and vitellogenm.
  • nfspN(H) 30t A nucleotide sequence for another nfspN nucleic acid molecule named nfspN(H) 30t is denoted as SEQ ID NO: 40.
  • a nucleotide sequence for another nfspN nucleic acid molecule named nfspN(I) 4QC is denoted as SEQ ID NO: 41.
  • a nucleotide sequence for another nfspN nucleic acid molecule named nfspN(J) bU is denoted as SEQ ID NO: 2.
  • a nucleotide sequence for another nfspN nucleic acid molecule named nfspN(K) 4/ is denoted as SEQ ID NO: 43.
  • a nucleotide sequence for another nfspN nucleic acid molecule named nfspN (L) 295 is denoted as SEQ ID NO:44.
  • nfspN(M) 37 ⁇ A nucleotide sequence for another nfspN nucleic acid molecule named nfspN(M) 37 ⁇ is denoted as SEQ ID NO: 45.
  • Nucleic acid sequence for portions of another nfspN nucleic acid molecule were obtained.
  • the nucleic acid molecule representing a 5' portion of nfspN(N) named nfspN(N) 252 is denoted as SEQ ID NO:46.
  • the nucleic acid molecule representing a 3' portion of nfspN(N) named nfspN (N) 6]3 is denoted as SEQ ID NO:47.
  • Nucleic acid sequence for portions of another nfspN nucleic acid molecule denoted herein as nfspN(O), were obtained.
  • nfspN(0) S38 The nucleic acid molecule representing a 5' portion of nfspN(O) named nfspN(0) S38 is denoted as SEQ ID NO: 48.
  • Translation of SEQ ID NO: 48 suggests that nucleic acid molecule nfspN (0) 538 encodes a non-full-length fspN(O) protein of about 178 ammo acids, referred to herein as PfspN(0) 178 , assuming the first residue spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 48.
  • the ammo acid sequence of PfspN(N) ⁇ ;a is denoted SEQ ID NO: 9.
  • nucleic acid molecule representing a 3' portion of nfspN(O) named nfspN(0) 4 is denoted as SEQ ID NO:50.
  • Translation of SEQ ID NO: 50 suggests that nucleic acid molecule nfspN(0) 4i , encodes a non-full-length fspN(O) protein of about 129 ammo acids, referred to herein as PfspN(O) :29 , assuming the first residue spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 50 and a stop codon spanning from about nucleotide 388 through about nucleotide 390 of SEQ ID NO: 50.
  • the ammo acid sequence of PfspN (0) 1 9 is denoted SEQ ID NO: 51
  • This example describes studies confirming the specificity of IgE enriched antiserum from CQQ2 to fspN protein.
  • a drop (about 100 pfu/drop) of each of the eighteen isolated phage clones was dropped onto each plate (18 phage clones/plate) .
  • the plates were incubated, filter lifted and the filters immunoscreened with IgE enriched antiserum from CQQ2, antiserum from a D.
  • This example describes the isolation of nucleic acid molecules encoding a fspG flea saliva protein. This example also describes expression of a fspG protein by bacteria.
  • a DNA probe labeled with ? P comprising nucleotides from nfspG4 610 (described m Example 2) was used to screen a flea salivary gland cDNA library (described m Example 6 of related PCT Publication No. WO 96/11,706) using standard hybridization techniques.
  • a clone was isolated havmg about a 595 nucleotide insert, referred to herein as nfspG5 5g having a nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO: 52.
  • nucleic acid molecule nfspG5 5g5 encodes a full-length flea salivary protein of about 90 ammo acids, referred to herein as PfspG5 90 , having ammo acid sequence SEQ ID NO: 53, assuming an open reading frame which the initiation codon spans from about nucleotide 46 through about nucleotide 48 of SEQ ID NO: 52 and the termination codon spans from about nucleotide 316 through about nucleotide 318 of SEQ ID NO: 52.
  • the complement of SEQ ID NO: 52 is represented herein by SEQ ID NO: 54.
  • the coding region encoding PfspG5 qo is represented by nucleic acid molecule nfspG5_ 7 , having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 55 and a complementary strand with nucleic acid sequence SEQ ID NO: 57.
  • the am o acid sequence of PfspG5 90 i.e., SEQ ID NO: 53 predicts that PfspG5 90 has an estimated molecular weight of about 9.6 kD and an estimated pi of about 9.28.
  • SEQ ID NO: 53 suggests the presence of a signal peptide encoded by a stretch of ammo acids spanning from about ammo acid 1 through about ammo acid 19.
  • the proposed mature protein denoted herein as PfsG5 71 , contains about 71 ammo acids which is represented herein as SEQ ID NO: 59.
  • the complement of SEQ ID NO: 58 is represented by
  • SEQ ID NO: 60 The ammo acid sequence of PfspG5 71 (i.e., SEQ ID NO: 60.
  • SEQ ID NO: 60 The ammo acid sequence of PfspG5 71 (i.e., SEQ ID NO: 60.
  • SEQ ID NO: 53 Comparison of ammo acid sequence SEQ ID NO: 53 with ammo acid sequences reported m GenBank indicates that SEQ ID NO: 53 showed the most homology, i.e., about 38% identity between SEQ ID NO: 53 and a Ctenocephalides feli s fl ea sal i vary pro tein FS-H precursor (GenBank accession
  • nfspG5 2]3 An about 213 bp nucleic acid molecule, referred to herein as nfspG5 2]3 (designed to encode an apparently mature flea salivary protein) was PCR amplified from nfspG5 595 using sense primer G7 having the nucleotide sequence 5' A GTG GAT CCG TCA AAA ATG GTC ACT G-3'
  • anti-sense primer G8 having the nucleotide sequence 5' CC GGA ATT CGG TTA TTC GCA ATA ACA GT-3'
  • nfspG5 (containing a £coRI shown m bold; denoted SEQ ID NO: 80) .
  • the resulting PCR product nfspG5 ?)3 was digested with BamHI and £coRI restriction endonucleases, gel purified, and subcloned into expression vector lambdaP R /T 7 or ⁇ /SlOHIS-RSET- A9, that had been digested with BamHI and £co.RI and dephosphorylated.
  • the resultant recombinant molecule referred to herein as pCro-nfspG5 2]3 , was transformed into E . coli BL-21 competent cells (available from Novagen, Madison, WI) to form recombinant cell E.
  • Example 8 The recombinant cell was cultured and induced as described in Example 11A of related PCT Publication No. WO 96/11,271. Immunoblot analysis of the proteins using a T7 antibody showed expression of an about 12 kD protein m the induced sample but not in the unmduced sample.
  • Example 8
  • This example describes the further sequencing of a nucleic acid sequence encoding a fspl flea saliva protein. This example also describes expression of a fspl protein by bacteria.
  • nucleic acid molecule denoted nfspl ⁇ , 73 described in Example 6 of related PCT Publication No. WO 96/11,706 was further sequenced using standard nucleotide sequencing methods.
  • a nucleic acid molecule was identified of about 1007 nucleotides, referred to herein as nfspl 1007/ the coding strand is denoted herein as SEQ ID NO: 61.
  • SEQ ID NO: 61 encodes a non-full- length flea salivary protein of about 155 ammo acids, referred to herein as Pfspl 155 , having ammo acid sequence SEQ ID NO: 62, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 61 and the termination codon spans from about nucleotide 466 through about nucleotide 468 of SEQ ID NO: 61.
  • the complement of SEQ ID NO: 61 is represented herein by SEQ ID NO: 63.
  • Flea salivary protein Pfspl 156 was produced m the following manner.
  • An about 474-bp nucleic acid molecule, referred to herein as nfspl 474 (designed to encode an apparently mature flea salivary protein) was PCR amplified from nfspl i0 ⁇ 7 using sense primer II havmg the nucleotide sequence 5' GCG CGG ATC CGC ATA TGG AAG ACA TCT GGA AAG TTA ATA
  • AAA AAT GTA CAT CAG-3 1 (containing an BamHI-site shown m bold as well as nucleic acid sequence encoding three am o acids, Glu-Asp-Isoleucme, shown in italics; denoted SEQ ID NO: 81) and anti-sense primer 12 having the nucleotide sequence 5' CCG GAA TTC TTA TTT ATT TTT TGG TCG ACA ATA ACA AAA GTT TCC-3' (containing a coRI shown m bold; denoted SEQ ID NO: 82) .
  • nfspl 474 which contained the nucleic acid sequences incorporated into primer II that encode three ammo acids, was digested with BamHI and £coRI restriction endonucleases, gel purified, and subcloned into expression vector lambdaP R /T or.-/S10HIS- RSET-A9, that had been digested with BamHI and Xbal and dephosphorylated.
  • the resultant recombinant molecule referred to herein as pCro-nfspI 474 , was transformed into E . coli BL-21 competent cells (available from Novagen, Madison, WI) to form recombinant cell E.
  • Example 9 The recombinant cell was cultured and protein production resolved using the methods described in Example 11A of related PCT Publication No. WO 96/11,271. Immunoblot analysis of the proteins using a T7 antibody showed expression of an about 30 kD protein m the induced sample but not in the unmduced sample.
  • Example 9 Immunoblot analysis of the proteins using a T7 antibody showed expression of an about 30 kD protein m the induced sample but not in the unmduced sample.
  • This example describes the isolation of nucleic acid molecules encoding a fspN flea saliva protein.
  • a DNA probe comprising nucleotides from nfspN(B) 6 (SEQ ID NO:52 of related PCT Publication No. WO 96/11,706) was labeled with i P and used to screen the flea salivary gland cDNA library using standard hybridization techniques.
  • a clone was isolated having about a 1205 nucleotide insert, referred to herein as nfspN5 12o;> havmg a nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO: 64.
  • SEQ ID NO: 64 suggests that nucleic ac d molecule nfspN5 120 encodes a non-full-length flea salivary protein of about 353 ammo acids, referred to herein as PfspN5 3b3 , having amino acid sequence SEQ ID NO: 65, assuming an open reading frame m which the initiation codon spans from about nucleotide 4 through about nucleotide 6 of SEQ ID NO: 64 and the termination codon spans from about nucleotide 1060 through about nucleotide 1062 of SEQ ID NO: 64.
  • the complement of SEQ ID NO: 64 is represented herem by SEQ ID NO: 66.
  • the coding region encoding PfspN5 353 is represented by nucleic acid molecule nfspN5 1059 , having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 67 and a complementary strand with nucleic acid sequence SEQ ID NO: 69.
  • the ammo acid sequence of PfspN5 3s (i.e., SEQ ID NO: 65) predicts that PfspN5 ⁇ has an estimated molecular weight of about 39.7 kD and an estimated pl of about 9.45.
  • SEQ ID NO: 65 Comparison of ammo acid sequence SEQ ID NO: 65 with ammo acid sequences reported m GenBank indicates that SEQ ID NO: 65 showed the most homology, i.e., about 32° identity between SEQ ID NO: 65 and a Human prostatic acid phosphatase precursor protein (GenBank accession P15309) . A GenBank homology search revealed no significant homology between SEQ ID NO: 64 and known nucleic acid sequences.
  • This example describes the isolation of nucleic acid molecules encoding a fspN flea saliva protein identified using IgE antibodies isolated from a dog having clinical flea allergy dermatitis.
  • a pool of sera (referred to herein as Pool #4) was collected from numerous known to have clinic flea allergy dermatitis (FAD) .
  • Pool #4 sera was used to identify flea saliva antigens that bind specifically to IgE antibodies m the FAD dog sera as follows.
  • Flea saliva extract was collected using the general methods described in Examples 1 and 2 of related PCT Publication No. WO 96/11,706, except a carboxymethyl cation exchange (CM) membrane (available from Schleicher and Scheull, Keene, NH) was used rather than a Durapore® membrane.
  • CM carboxymethyl cation exchange
  • flea saliva extract was eluted from the membrane by contacting the membrane m an extraction buffer of 2.5 M NaCl, 5° 0 isopropyl alcohol (IPA) and 20 mM Tris, pH 8.0. The membrane was eluted overnight at room temperature.
  • the flea saliva extract was resolved by high pressure liquid chromatography (HPLC) using the method generally described in Example 2 of related PCT Publication No. WO 96/11,706. Proteins contained the HPLC fractions were resolved on a 16°o Tris-glycme SDS PAGE gel. Proteins on the gel were then blotted to an Immobilon PTM filter (available from Millipore Co., Bedford, MA) using standard Western Blot techniques.
  • HPLC high pressure liquid chromatography
  • IgE antibodies bound to protein on the blot was then detected as follows.
  • the blot was first incubated with about a 1:200 dilution of Pool #4 sera using standard antibody hybridization techniques, washed, and then incubated with about a 1:500 dilution of a 145 ⁇ g/milliliter solution of biotmylated human Fc R alpha chain protein using standard Western Blot techniques. Following washing, the blot was incubated with about a 1:5,000 dilution of streptavid conjugated to alkaline phosphatase (available from Sigma, St. Louis, MO) .
  • BCIP/NBT substrate available from Gibco BRL, Gaithersburg, MD
  • BCIP/NBT substrate available from Gibco BRL, Gaithersburg, MD
  • Protein bands were detected m samples containing Fractions 34, 37, 38, 47, 49, 51, 52 and 53.
  • Ammo (N-) terminal ammo acid sequencing analysis was performed on protein contained in the about 40 kD protein band identified m the sample containing Fraction 52, using standard procedures known to those in the art (see, for example, Geisow et al .
  • Synthetic oligonucleotide primers were designed using SEQ ID NO: 70 and used to isolate a nucleic acid molecule encoding SEQ ID NO: 70 as follows.
  • Sense primer 1 having the nucleotide sequence 5' AAA TTT GTA(T) TTT GTA(T) ATG GTA(T) AAA GGA(T) CCA(T) GAT CAT GAA GC -3' (denoted SEQ ID NO: 83) was used m combination with the M13 forward universal standard primer 5' GTAAAACGACGGCCAGT 3' (denoted SEQ ID NO: 84) to produce a PCR product from the a flea salivary gland cDNA library described above in Example 9. PCR amplification was conducted using standard techniques.
  • the resulting PCR amplification product was a fragment of about 406 nucleotides, denoted herein as nfspN6 40b .
  • the PCR product was cloned into the InVitrogen, Corp., TATM cloning vector (procedures provided by InVitrogen, Corp.) and subjected to DNA sequence analysis using standard techniques.
  • nucleic acid sequence of the coding strand of nfspN6 40o is denoted herein as SEQ ID NO: 71.
  • Translation of SEQ ID NO: 71 suggests that nucleic acid molecule nfspN6 40t encodes a non-full-length flea salivary protein of about 135 ammo acids, referred to herem as PfspN6 13 , having amino acid sequence SEQ ID NO: 72, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 71 and the last codon spans from about nucleotide 403 through about nucleotide 405 of SEQ ID NO: 71.
  • SEQ ID NO: 73 The complement of SEQ ID NO: 71 is represented herein by SEQ ID NO: 73.
  • a GenBank homology search revealed no significant homology between amino acid sequence SEQ ID NO:72 and nucleic acid sequence SEQ ID NO: 71 and known amino acid sequences or nucleic acid sequences, respectively.
  • Example 11 This example describes the isolation of nucleic acid molecules encoding a fspj flea saliva protein.
  • oligonucleotide primers were designed from the ammo acid sequence deduced for fspJ (described m Example 4 of related PCT Publication No.WO 96/11,706) and were used to isolate a fspJ nucleic acid molecule as follows. Two synthetic oligonucleotides were synthesized that corresponded to the region of fspJ spanning from about residues 7 through about 26 of SEQ ID NO: 8 of related PCT Publication No.WO 96/11,706. Primer 1, a "sense" primer corresponding to ammo acid residues fro about residue 7 to about 16 of SEQ ID NO: 8 of related PCT Publication No.
  • O 96/11,706 has the nucleotide sequence 5' CAT GAA CCA(T) GGA(T) AAT ACA(T) CGA(T) AAA(G) ATA(C/T) A(C)G 3' (denoted herein as SEQ ID NO: 84) .
  • Primer 2 a "sense" primer corresponding to ammo acid residues form about residue 17 through about 26 of SEQ ID NO: 8 of related PCT Publication No. WO 96/11,706, has the nucleic acid sequence 5' GAA GTA(T) ATG GAC(T) AAA TTA(G) AGA(G) CAA(G) GC -3' (denoted herein as SEQ ID NO: 86) .
  • Example 9 was conducted using standard techniques. PCR amplification products were generated using a combination of Primer 1 and M13 primer (denoted SEQ ID NO: 85) . The resultant PCR products were used for a nested PCR amplification using Primer 2 and the T7 standard primer 5' GTA ATA CGA CTC ACT ATA TAG GGC 3' (denoted SEQ ID NO: 88) . The resultant PCR product, a fragment of about 420 nucleotides, denoted herein as nfspJ 420 .
  • the PCR product was cloned into the InVitrogen, Corp., TA 11*1 cloning vector (procedures provided by InVitrogen, Corp.) and subjected to DNA sequence analysis using standard techniques.
  • the nucleic acid sequence of the coding strand of nfspJ 4 - 0 is denoted herein as SEQ ID NO: 74.
  • SEQ ID NO: 74 suggests that nucleic acid molecule nfspJ 42 ⁇ encodes a non-full-length flea salivary protein of about 72 ammo acids, referred to herein as PfspJ ⁇ , having amino acid sequence SEQ ID NO: 75, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:74 and the last codon spans from about nucleotide 214 through about nucleotide 216 of SEQ ID NO: 74.
  • the complement of SEQ ID NO: 74 is represented herein by SEQ ID NO: 76.
  • GenBank homology search revealed no significant homology between amino acid sequence SEQ ID NO:75 and nucleic acid sequence SEQ ID NO: 74 and known ammo acid sequences or nucleic acid sequences, respectively.
  • This example describes the ammo acid sequence analysis of an isolated and HPLC purified fspN7 flea saliva protein. Fractions of flea saliva proteins described above m
  • Example 10 were tested for the ability to stimulate T cell clones that respond specifically to the flea saliva extract described m Example 10 (FS-specific T cells) .
  • T cell activation were performed using standard methods such as those described in Current Protocols m Immunol ogy, Vol. 1, Chapter 3 [3.13.2], ed. J.E. Coligan et al . , pub. Wiley Interscience, 1993. Briefly, about 10' FS-1-specxfic I cells (clone CP02-7; isolated from dog CP02 described m Example 8 of related PCT Patent Publication No.
  • WO 96/11,271 were added to individual wells of a 96 well tissue culture plate, in the presence of about 2 x 104 autologous antigen presenting cells (isolated by ficoll gradient from dog CP02) and about 100 units/milliliter of recombinant human mterleukm-2 (Proleukm®; available from Chiron Inc., Emeryville, CA) . About 1 microliter of each fraction of protein resolved by HPLC was to added to each well m triplicate. The cells were incubated for about 4 to about 6 days. About 16 hours prior to harvesting, about 1 ⁇ Ci of tritiated thymid e (available from Amersham Inc., Arlington Heights, IL) was added to each well.
  • tritiated thymid e available from Amersham Inc., Arlington Heights, IL
  • Fraction 51 protein contained m a HPLC fraction containing fspN protein (Fraction 51) stimulated the FS- specific T cells.
  • Ammo (N-) terminal amino acid sequencing analysis was performed on protein contained in Fraction 51 using standard procedures known to those in the art (see, for example, Geisow et al . , ibid. ; Hewick et al . , 1981, ibid. ) .
  • the N-termmal partial ammo acid sequence of the band was determined to be Asn Asp Lys Leu Gin Phe Val Phe Val Met Ala Arg Gly Pro Asp His Glu Ala Cys Asn Tyr Pro Gly Gly Pro (denoted herein as SEQ ID NO:78) .
  • SEQ ID NO:78 The N-termmal partial ammo acid sequence of the band was determined to be Asn Asp Lys Leu Gin Phe Val Phe Val Met Ala Arg Gly Pro Asp His Glu Ala Cys Asn Tyr Pro Gly Gly Pro (denoted herein as SEQ ID NO:78) .
  • This example describes the ammo acid sequence analysis of an isolated and HPLC purified fspM2 flea saliva protein.
  • Proteins contained withm Fraction 47 described above m Example 10 were resolved on a 16% Tris-glycme SDS PAGE gel. A major band at about 34 kD was identified. Ammo (N-) terminal ammo acid sequencing analysis was performed on protein contained in the about 34 kD using standard procedures known to those m the art (see, for example, Geisow et al., ibid. ; Hewick et al., 1981, ibid. ) . The N- termmal partial ammo acid sequence of the band was determined to be Tyr Phe Asn Lys leu Val Gin Ser Trp Thr Glu Pro Met Val Phe Lys Tyr Pro Tyr (denoted herein as SEQ ID NO:87) .
  • ADDRESSEE SHERIDAN ROSS P.C.
  • MOLECULE TYPE protein
  • XI SEQUENCE DESCRIPTION' SEQ ID NO: 6:
  • AAA ATG GTC ACT GAA AAG TGT AAG TCA GGT GGA AAT AAT CCA AGT ACA 152 Lys Met Val Thr Glu Lys Cys Lys Ser Gly Gly Asn Asn Pro Ser Thr
  • MOLECULE TYPE protein ( XI ) SEQUENCE DESCRI PTION : SEQ ID NO : 14 :
  • MOLECULE TYPE protein
  • GGT TCT ACT GAC GGA AAT GGA AAT TTT ATA AGT ACT TTT AGT GAT CAT 383 Gly Ser Thr Asp Gly Asn Gly Asn Phe He Ser Thr Phe Ser Asp His 115 120 125
  • CAATATGCCC AAAATATAAC TTATAATTCA AATATCAGTC CTGAAGTGAT TGGATTCCGA 480 GAACATTATG TTGCGGAACA GCCTTCTGGT GACGTGCTTC ACAAAAGTCA TTTAACAGAA 540
  • ATCACACCTC AACTTGACAG CTTACGATCA CGAGATATAG TAATTAAGGG AGAATTACTA 960
  • ACG AAA ATG GAT ACT GTT GTA CAA GAA ATT GAA AGC AAA GAA TCT GAG 1200 Thr Lys Met Asp Thr Val Val Gin Glu He Glu Ser Lys Glu Ser Glu 385 390 395 400
  • AAG AAA GCC AAA ACA TTG CCA CTT GAA GCT CCT AGG AGC GCT ACT GAA 1248 Lys Lys Ala Lys Thr Leu Pro Leu Glu Ala Pro Arg Ser Ala Thr Glu 405 410 415 ACT CAA GAA TTA GAT GTA AGG AAA GAA AGA GGA GAG ATT TTA ATT GAC 1296 Thr Gin Glu Leu Asp Val Arg Lys Glu Arg Gly Glu He Leu He Asp 420 425 430
  • MOLECULE TYPE protein
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:30:

Abstract

La présente invention concerne un nouveau produit et un nouveau procédé pour isoler des protéines de salive d'ectoparasite, ainsi qu'un nouveau produit et un nouveau procédé pour détecter et/ou traiter l'eczéma allergique chez un animal. L'invention concerne également des protéines de salive d'ectoparasite, des molécules d'acide nucléique présentant des séquences codant pour ces protéines, et des anticorps dirigés contre ces dernières. Elle concerne d'autre part des procédés pour obtenir de telles protéines et pour les utiliser en vue d'identifier des animaux prédisposés à l'eczéma allergique ou en souffrant. La présente invention a trait par ailleurs à des compositions thérapeutiques comprenant ces protéines et à leur utilisation pour traiter des animaux prédisposés à l'eczéma allergique ou en souffrant.
PCT/US1997/005959 1994-10-07 1997-04-10 Nouvelles proteines de salive d'ectoparasite et appareil pour les recueillir WO1997037676A1 (fr)

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US09/171,156 US6368846B1 (en) 1996-04-10 1997-04-10 Ectoparasite saliva proteins and apparatus to collect such proteins
JP53649997A JP4694657B2 (ja) 1996-04-10 1997-04-10 新規外部寄生生物唾液タンパク質および同タンパク質を収集する装置
CA2250835A CA2250835C (fr) 1996-04-10 1997-04-10 Nouvelles proteines de salive d'ectoparasite et appareil pour les recueillir
AU24531/97A AU719742B2 (en) 1996-04-10 1997-04-10 Novel ectoparasite saliva proteins and apparatus to collect such proteins
EP97920304A EP0939642A4 (fr) 1996-04-10 1997-04-10 Nouvelles proteines de salive d'ectoparasite et appareil pour les recueillir
AU49849/97A AU4984997A (en) 1997-04-10 1997-10-15 Novel ectoparasite saliva proteins and apparatus to collect such proteins
US08/981,799 US6576238B1 (en) 1994-10-07 1997-10-15 Ectoparasite saliva proteins and apparatus to collect such proteins
PCT/US1997/018669 WO1998045408A2 (fr) 1996-04-10 1997-10-15 Nouvelles proteines de salive d'ectoparasite et appareil permettant de recueillir ces proteines
US09/004,730 US6485968B1 (en) 1994-10-07 1998-01-08 Ectoparasite saliva proteins and apparatus to collect such proteins
US11/694,771 US20080045693A1 (en) 1996-04-10 2007-03-30 Novel flea saliva protein

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Application Number Priority Date Filing Date Title
US08/630,822 1996-04-10
US08/630,822 US5840695A (en) 1994-10-07 1996-04-10 Ectoparasite saliva proteins and apparatus to collect such proteins

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US09/171,156 A-371-Of-International US6368846B1 (en) 1996-04-10 1997-04-10 Ectoparasite saliva proteins and apparatus to collect such proteins
US08/981,799 Continuation-In-Part US6576238B1 (en) 1994-10-07 1997-10-15 Ectoparasite saliva proteins and apparatus to collect such proteins
PCT/US1997/018669 Continuation-In-Part WO1998045408A2 (fr) 1994-10-07 1997-10-15 Nouvelles proteines de salive d'ectoparasite et appareil permettant de recueillir ces proteines
US10/071,751 Division US20020142352A1 (en) 1996-04-10 2002-02-07 Novel ectoparasite salvia proteins and apparatus to collect such proteins

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US7033790B2 (en) 2001-04-03 2006-04-25 Curagen Corporation Proteins and nucleic acids encoding same
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US6485968B1 (en) * 1994-10-07 2002-11-26 Heska Corporation Ectoparasite saliva proteins and apparatus to collect such proteins
US20020169104A1 (en) * 1997-05-01 2002-11-14 Glenn Frank Novel ectoparasite saliva proteins and apparatus to collect such proteins
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WO2000061621A2 (fr) * 1999-04-09 2000-10-19 Heska Corporation Molecules d'acides nucleiques et proteines issues de la tete, de la moelle epiniere, de l'intestin posterieur et du tube de malpighi de puces et utilisations correspondantes
WO2000061621A3 (fr) * 1999-04-09 2001-07-05 Heska Corp Molecules d'acides nucleiques et proteines issues de la tete, de la moelle epiniere, de l'intestin posterieur et du tube de malpighi de puces et utilisations correspondantes
JP2003501010A (ja) * 1999-04-09 2003-01-14 ヘスカ コーポレイション ノミ頭部、神経索、後腸及びマルピーギ管の核酸分子、蛋白質及びそれらの使用方法
EP1710252A2 (fr) * 1999-04-09 2006-10-11 Heska Corporation Molécules d'acides nucléiques et protéines issues de la tête, de la moelle épinière et du tube de Malpighi de puces et les utilisations correspondantes
EP1710252A3 (fr) * 1999-04-09 2006-12-27 Heska Corporation Molécules d'acides nucléiques et protéines issues de la tête, de la moelle épinière et du tube de Malpighi de puces et les utilisations correspondantes.
EP1947112A3 (fr) * 1999-04-09 2008-08-06 Heska Corporation Molécules d'acides nucléiques de tête, cordon nerveux, intestin postérieur et tube de Malpighi de puce et leurs utilisations
US7033790B2 (en) 2001-04-03 2006-04-25 Curagen Corporation Proteins and nucleic acids encoding same
US8349333B2 (en) * 2005-12-23 2013-01-08 China Agricultural University Allergy inhibitor compositions and kits and methods of using the same
US8795675B2 (en) 2005-12-23 2014-08-05 Bin Wang Allergy inhibitor compositions and kits and methods of using the same
US9962437B2 (en) 2005-12-23 2018-05-08 Bin Wang Allergy inhibitor compositions and kits and methods of using the same

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EP0939642A4 (fr) 2003-06-18
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AU2453197A (en) 1997-10-29
ZA973023B (en) 1997-11-04
CA2250835C (fr) 2013-07-23
AU719742B2 (en) 2000-05-18
JP2000509972A (ja) 2000-08-08
CA2250835A1 (fr) 1997-10-16
AR008588A1 (es) 2000-02-09
IL120620A0 (en) 1997-08-14
US5932470A (en) 1999-08-03
JP4694657B2 (ja) 2011-06-08
US5840695A (en) 1998-11-24

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